Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Applied Biosystems7900HT Fast Real-Time PCR Systemand SDS Enterprise DatabaseUser Guide

© Copyright 2004, Applied Biosystems. All rights reserved.For Research Use Only. Not for use in diagnostic procedures.Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. Thisdocument is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, orconsequential damages in connection with or arising from the use of this document.AUTHORIZED THERMAL CYCLER: This Applied Biosystems 7900HT Fast Real-Time PCR System Base Unit, Serial No___________, in combination with itsimmediately attached sample block modules, comprise an Authorized Thermal Cycler. The purchase price of this Base Unit includes the up-front fee component of alicense under United States Patent Nos. 4,683,195, 4,683,202 and 4,965,188, owned by Roche Molecular Systems, Inc., and under corresponding claims in patentsoutside the United States, owned by F. Hoffmann-La Roche Ltd, covering the Polymerase Chain Reaction (“PCR”) process to practice the PCR process for internalresearch and development using this instrument. The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasingAuthorized Reagents. This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems. Its use withAuthorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of this product does not itselfconvey to the purchaser a complete license or right to perform the PCR process. Further information on purchasing licenses to practice the PCR process may beobtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404.DISCLAIMER OF LICENSE: No rights for any application, including any in vitro diagnostic application, are conveyed expressly, by implication or by estoppelunder any patent or patent applications claiming homogeneous or real-time detection methods, including patents covering such methods used in conjunction with thePCR process or other amplification processes. The 5' nuclease detection assay and certain other homogeneous or real-time amplification and detection methods arecovered by United States Patent Nos. 5,210,015, 5,487,972, 5,804,375 and 5,994,056, owned by Roche Molecular Systems, Inc.; by corresponding patents and patentapplications outside the United States, owned by F. Hoffmann-La Roche Ltd; and by United States Patent Nos. 5,538,848 and 6,030,787, and corresponding patentsand patent applications outside the United States, owned by Applera Corporation. Purchase of this instrument conveys no license or right under the foregoing patents.Use of these and other patented processes in conjunction with the PCR process requires a license. For information on obtaining licenses, contact the Director ofLicensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, or The Licensing Department, Roche Molecular Systems, Inc., 1145Atlantic Avenue, Alameda, California, 94501NOTICE TO PURCHASER: PLEASE REFER TO THE SDS ENTERPRISE DATABASE USER'S MANUAL FOR LIMITED LABEL LICENSE ORDISCLAIMER INFORMATION.JpegEncoder Licensing StatementThe JpegEncoder and its associated classes are Copyright © 1998, James R. Weeks and BioElectroMech. This software is based in part on the work of the IndependentJPEG Group.Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:1. Redistributions of source code must retain the above copyright notice, this list of conditions, all files included with the source code, and the following disclaimer.2. Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or othermaterials provided with the distribution.THIS SOFTWARE (JpegEncoder) IS PROVIDED BY THE AUTHOR AND CONTRIBUTORS ``AS IS'' AND ANY EXPRESS OR IMPLIED WARRANTIES,INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE AREDISCLAIMED. IN NO EVENT SHALL THE AUTHOR OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL,EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES;LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER INCONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THISSOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.TRADEMARKS:ABI PRISM, Applied Biosystems, MicroAmp, Primer Express, and VIC are registered trademarks and AB (Design), ABI PRISM, Applera, FAM, JOE, NED, ROX,TAMRA, and TET are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.AmpErase, AmpliTaq Gold, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.SYBR is a registered trademark of Molecular Probes, Inc.Zymark is a registered trademark of Zymark Corporation.Windows and Windows NT are registered trademarks of Microsoft Corporation.All other trademarks are the sole property of their respective owners.Part Number 4351684 Rev. A09/2004DRAFTSeptember 1, 2004 11:38 am, 4351684A_Title.fm

ContentsPrefaceHow to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiHow to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiiHow to Obtain Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiiSafety and EMC Compliance InformationSafety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xivSymbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvSafety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviGeneral Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviiChemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviiiChemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xixElectrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxPhysical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiBiological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiLaser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiBar Code Scanner Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiiWorkstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiiiSafety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . . . xxiiiChapter 1Product OverviewSystem Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2Section 1.1 Getting to Know the Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-37900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9Instrument Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10Section 1.2 Getting to Know the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fmiii

Managing Sequence Detection System Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17Section 1.3 SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-21About the SDS Enterprise Database Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22About the SDS Enterprise Database Software Suite . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25Database Modules for Large-Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25Database Management Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28Chapter 2Getting StartedGetting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4Using the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-11Lesson 1: Using Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12Lesson 2: Viewing and Resizing Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-21Lesson 3: Using the Plate Grid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-23Lesson 4: Using the Hand-Held Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . 2-27Lesson 5: Using Contextual Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28Lesson 6: Using Keyboard Shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-28Using SDS Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29Chapter 3Preparing a RunNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5Quick Review: Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9Step 2 – Applying Detectors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11Step 3 – Configuring the Plate Document with Tasks . . . . . . . . . . . . . . . . . . . . . . . . . 3-16Step 4 – Setting the Passive Reference and Omitting Wells . . . . . . . . . . . . . . . . . . . . 3-18Step 5 – Programming the Plate Document Method . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19Step 6 – Saving the Plate Document as a Template . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24Step 7 – Creating a Plate Document from the Template . . . . . . . . . . . . . . . . . . . . . . . 3-26Step 8 – Applying Sample and Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-28Step 9 – Running the Plate on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . 3-29ivApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fm

Chapter 4Operating the InstrumentNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4Section 4.1 Consumable Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8Preparing TaqMan Low Density Arrays for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10About the Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10About the Centrifuge System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12About the Sealer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14Loading the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14Centrifuging the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-16Sealing the TaqMan Low Density Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-19Section 4.2 Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-25After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29Section 4.3 Automated Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-31Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . 4-32Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-34Adding a Plate Document to the Plate Queue (Using the SDS Software) . . . . . . 4-35Creating Plate Documents Using the Template Batch Utility . . . . . . . . . . . . . . . . 4-36Starting and Configuring the Automation Controller Software for Operation . . . . 4-39Running Plates Using the Automation Controller Software . . . . . . . . . . . . . . . . . . . . 4-41Loading Plates onto the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-41Running the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-43After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44Chapter 5Analyzing End-Point DataEnd-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fmv

Chapter 6Analyzing Real-Time DataNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16Algorithmic Manipulation of Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17Automatic Outlier Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19Essential Experimental Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26About the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26Specifying the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-29Analyzing the Study Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-30Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Determining T m Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52viApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fm

Chapter 7Maintaining the InstrumentNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4Section 7.1 Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-5Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6Changing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Preparing the Background Plate or TaqMan Low Density Array . . . . . . . . . . . . . 7-17Creating a Plate Document for the Background Run . . . . . . . . . . . . . . . . . . . . . . 7-17Running the Prepared Background Plate or TaqMan Low Density Array . . . . . . 7-18Analyzing the Background Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-19Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Preparing the Pure Dye Plates or Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-22Preparing a Plate Document for a Pure Dye Plate or Card . . . . . . . . . . . . . . . . . 7-22Running the Prepared Pure Dye Plate or Card . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24Analyzing the Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35Automation Accessory Components and Stack Positions . . . . . . . . . . . . . . . . . . . . . 7-36Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . 7-37Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . . . . . 7-53General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54Maintaining the SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55Chapter 8TroubleshootingTroubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9Pure Dye Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11Real-Time Runs (Quantitative PCR and Dissociation Curves) . . . . . . . . . . . . . . . . . . 8-12End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . . . . 8-17TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fmvii

Appendix A Software ReferenceImporting Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-41. File Version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5Plate Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-52. Plate Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-53. Plate ID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-5Detector Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-64. Number of Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-65. Detectors List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-66. Detectors List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7Marker Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-87. Number of Markers (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . . A-88. Markers List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . A-89. Markers List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-9Well-Detector Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1010. Well-Detector List Header . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1011. Well-Detector Definition List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10Well-Marker Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1212. Well-Marker List Header (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . . . A-1213. Well-Marker Definition List (Allelic Discrimination Only) . . . . . . . . . . . . . . . . . A-12Example Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-14Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16Operating the SDS Software from a Command Line . . . . . . . . . . . . . . . . . . . . . . . . . . A-18Using the Search Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-20Connecting SDS Software to the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22Appendix B Designing TaqMan Reagent-Based AssaysAssay Development Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2Design Tips for Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5Design Tips for Quantitative PCR Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6Appendix C Kits, Reagents, and ConsumablesInterchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . . . . . C-2Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5TaqMan Pre-Developed Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6viiiApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fm

Appendix D Theory of OperationFluorescent-Based Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2Real-Time Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-4Comparative C T Method of Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . D-8Appendix E Limited Warranty StatementBibliographyGlossaryIndexApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fmix

xApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fm

PrefaceHow to Use This GuidePurpose of ThisGuideAudienceAssumptionsText ConventionsUser AttentionWordsSafety AlertWordsThe Applied Biosystems 7900HT Fast Real-Time PCR System and SDS EnterpriseDatabase User Guide describes how to prepare, maintain, and troubleshoot theApplied Biosystems 7900HT Fast Real-Time PCR System instrument, Zymark ®Twister Microplate Handler, and SDS Enterprise Database.This guide is written for technicians, scientists, and researchers of all skill levels whowill use and maintain 7900HT instruments with or without the SDS EnterpriseDatabase.This guide assumes that a Applied Biosystems technical representative has installedyour 7900HT instrument, Plate Handler, and SDS Enterprise Database and that youare familiar with:• Basic Microsoft ® Windows ® operations such as using the mouse, choosingcommands, working with windows, and using the hierarchical file system• Electronic storage devices (computer hard drives) and data files• Assay preparation and basic laboratory techniquesThis guide uses the following conventions:• Bold indicates user action. For example:Enter 0, then press Enter for each of the remaining fields.• Italics indicates new or important words and is used for emphasis. For example:Before performing a pure dye calibration, always perform a background run.• A right arrow bracket (>) separates successive commands you select from adrop-down or shortcut menu. For example:Select File > Open > Spot Set.Two user attention words appear in Applied Biosystems user documentation. Eachword implies a particular level of observation or action as described below:Note: Provides information that may be of interest or help but is not critical to theuse of the product.IMPORTANT! Provides information that is necessary for proper instrumentoperation, accurate chemistry kit use, or safe use of a chemical.Safety alert words also appear in user documentation. For more information,see “Safety Alert Words” on page xiv.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Preface.fmxi

PrefaceHow to Obtain More InformationRelatedDocumentationSend Us YourCommentsSee the following related documents for more information on the topics in this guide:• Applied Biosystems 7900HT Fast Real-Time PCR System Quick Starts –Provide brief, procedures for performing application-specific tasks on the7900HT instrument.• Sequence Detections Systems Software Online Help – Describes the SequenceDetection Systems (SDS) Software and provides procedures for common tasks.• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-TimePCR System Administrators Guide – Provides information for databaseadministrators who will be maintaining the SDS Enterprise Database, andprovides information for systems integrators who will be working with the SDSEnterprise Database API.Note: For additional documentation, see “How to Obtain Services and Support” onpage xii.Applied Biosystems welcomes your comments and suggestions for improving itsuser documents. You can e-mail your comments to:techpubs@appliedbiosystems.comHow to Obtain Services and SupportTo contact Applied Biosystems Technical Support from North America bytelephone, call 1.800.899.5858.For the latest services and support information for all locations, go tohttp://www.appliedbiosystems.com, then click the link for Services and Support.At the Services and Support page, you can:• Search through frequently asked questions (FAQs)• Submit a question directly to Technical Support• Order Applied Biosystems user documents, MSDSs, certificates of analysis,and other related documents• Download PDF documents• Obtain information about customer training• Download software updates and patchesIn addition, the Services and Support page provides access to worldwide telephoneand fax numbers to contact Applied Biosystems Technical Support and Salesfacilities.xiiApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Preface.fm

Safety and EMC Compliance InformationThis section includes the following topics:Safety Conventions Used in This Document. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xivSymbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xvSafety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviGeneral Instrument Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviiChemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviiiChemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xixElectrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxPhysical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiBiological Hazard Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiLaser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiBar Code Scanner Laser Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiiWorkstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiiiSafety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . xxiiiApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxiii

Safety and EMC Compliance InformationSafety Conventions Used in This DocumentSafety AlertWordsFour safety alert words appear in Applied Biosystems user documentation at pointsin the document where you need to be aware of relevant hazards. Each alertword–IMPORTANT, CAUTION, WARNING, DANGER–implies a particularlevel of observation or action, as defined below:DefinitionsIMPORTANT! – Indicates information that is necessary for proper instrumentoperation, accurate chemistry kit use, or safe use of a chemical.– Indicates a potentially hazardous situation that, if not avoided,may result in minor or moderate injury. It may also be used to alert against unsafepractices.– Indicates a potentially hazardous situation that, if not avoided,could result in death or serious injury.– Indicates an imminently hazardous situation that, if not avoided,will result in death or serious injury. This signal word is to be limited to the mostextreme situations.Except for IMPORTANTs, each safety alert word in an Applied Biosystemsdocument appears with an open triangle figure that contains a hazard symbol. Thesehazard symbols are identical to the hazard icons that are affixed to AppliedBiosystems instruments (see “Safety Symbols” on page xv).ExamplesThe following examples show the use of safety alert words:IMPORTANT! You must create a separate a Sample Entry Spreadsheet for each96-well microtiter plate.The lamp is extremely hot. Do not touch the lamp until it hascooled to room temperature.CHEMICAL HAZARD. Formamide. Exposure causes eye,skin, and respiratory tract irritation. It is a possible developmental and birth defecthazard. Read the MSDS, and follow the handling instructions. Wear appropriateprotective eyewear, clothing, and gloves.ELECTRICAL HAZARD. Failure to ground the instrumentproperly can lead to an electrical shock. Ground the instrument according to theprovided instructions.xivApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

Symbols on InstrumentsSymbols on InstrumentsElectricalSymbols onInstrumentsThe following table describes the electrical symbols that may be displayed onApplied Biosystems instruments.Symbol Description Symbol DescriptionIndicates the On position of themain power switch.Indicates the Off position of themain power switch.Indicates a protectivegrounding terminal that mustbe connected to earth groundbefore any other electricalconnections are made to theinstrument.Indicates the On/Off position ofa push-push main powerswitch.Indicates a terminal that maybe connected to the signalground reference of anotherinstrument. This is not aprotected ground terminal.Indicates a terminal that canreceive or supply alternatingcurrent or voltage.Indicates a terminal that canreceive or supply alternating ordirect current or voltage.Safety SymbolsThe following table describes the safety symbols that may be displayed onApplied Biosystems instruments. Each symbol may appear by itself or in combinationwith text that explains the relevant hazard (see “Safety Labels on Instruments” onpage xvi). These safety symbols may also appear next to DANGERS, WARNINGS,and CAUTIONS that occur in the text of this and other product-support documents.Symbol Description Symbol DescriptionIndicates that you shouldconsult the manual for furtherinformation and to proceedwith appropriate caution.Indicates the presence of anelectrical shock hazard and toproceed with appropriatecaution.Indicates the presence of alaser inside the instrument andto proceed with appropriatecaution.Indicates the presence ofmoving parts and to proceedwith appropriate caution.Indicates the presence of a hotsurface or otherhigh-temperature hazard andto proceed with appropriatecaution.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxv

Safety and EMC Compliance InformationSafety Labels on InstrumentsThe following CAUTION, WARNING, and DANGER statements may be displayedon Applied Biosystems instruments in combination with the safety symbolsdescribed in the preceding section.EnglishCAUTION Hazardous chemicals. Read the Material SafetyData Sheets (MSDSs) before handling.CAUTION Hazardous waste. Read the waste profile (if any)in the site preparation guide for this instrument beforehandling or disposal.CAUTION Hazardous waste. Refer to MSDS(s) and localregulations for handling and disposal.WARNING Hot lamp.WARNING Hot. Replace lamp with an Applied Biosystemslamp.CAUTION Hot surface.DANGER High voltage.WARNING To reduce the chance of electrical shock, do notremove covers that require tool access. No user-serviceableparts are inside. Refer servicing to Applied Biosystemsqualified service personnel.DANGER Class 3B laser radiation present when open andinterlock defeated. Avoid direct exposure to laser beam.DANGER Class 3B laser radiation when open. Avoid directexposure to laser beam.DANGER Class 3B laser radiation present when open andinterlock defeated. Do not stare directly into the beamDANGER Class 3B laser radiation present when open. Donot stare directly into the beam.DANGER Class 3B LED when open and interlock defeated.Do not stare directly into the beam.DANGER Class 3B LED when open. Do not stare directlyinto the beam.CAUTION Moving parts.FrancaisATTENTION Produits chimiques dangeureux. Lire les fichestechniques de sûreté de matériels avant la manipulation desproduits.ATTENTION Déchets dangereux. Lire les renseignementssur les déchets avant de les manipuler ou de les éliminer.ATTENTION Déchets dangereux. Lire les fiches techniquesde sûreté de matériels et la régulation locale associées à lamanipulation et l'élimination des déchets.AVERTISSEMENT Lampe brûlante.AVERTISSEMENT Composants brûlants. Remplacer lalampe par une lampe Applied Biosystems.ATTENTION Surface brûlante.DANGER Haute tension.AVERTISSEMENT Pour éviter les risques d'électrocution, nepas retirer les capots dont l'ouverture nécessite l'utilisationd'outils. L’instrument ne contient aucune pièce réparable parl’utilisateur. Toute intervention doit être effectuée par lepersonnel de service qualifié de Applied Biosystems.DANGER Class 3B rayonnement laser en cas d’ouverture etd’une neutralisation des dispositifs de sécurité. Eviter touteexposition directe avec le faisceau.DANGER Class 3B rayonnement laser en cas d’ouverture.Eviter toute exposition directe avec le faisceau.DANGER de Class 3B rayonnement laser en cas d'ouvertureet d'une neutralisation des dispositifs de securite. Evitertoute exposition directe avec le faisceau.DANGER de Class 3B rayonnement laser en casd'ouverture. Eviter toute exposition directe avec le faisceau.DANGER de Class 3B LED en cas d'ouverture et d'uneneutralisation des dispositifs de securite. Eviter touteexposition directe avec le faisceau.DANGER de Class 3B LED en cas d'ouverture. Eviter touteexposition directe avec le faisceau.ATTENTION Parties mobiles.xviApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

General Instrument SafetyGeneral Instrument SafetyPHYSICAL INJURY HAZARD. Use this product only asspecified in this document. Using this instrument in a manner not specified byApplied Biosystems may result in personal injury or damage to the instrument.Moving andLifting theInstrumentMoving andLifting Stand-Alone Computersand MonitorsOperating theInstrumentPHYSICAL INJURY HAZARD. The instrument is to be movedand positioned only by the personnel or vendor specified in the applicable sitepreparation guide. If you decide to lift or move the instrument after it has beeninstalled, do not attempt to lift or move the instrument without the assistance ofothers, the use of appropriate moving equipment, and proper lifting techniques.Improper lifting can cause painful and permanent back injury. Depending on theweight, moving or lifting an instrument may require two or more persons.Do not attempt to lift or move the computer or the monitorwithout the assistance of others. Depending on the weight of the computer and/or themonitor, moving them may require two or more people.Things to consider before lifting the computer and/or the monitor:• Make sure that you have a secure, comfortable grip on the computer or themonitor when lifting.• Make sure that the path from where the object is to where it is being moved isclear of obstructions.• Do not lift an object and twist your torso at the same time.• Keep your spine in a good neutral position while lifting with your legs.• Participants should coordinate lift and move intentions with each other beforelifting and carrying.• Instead of lifting the object from the packing box, carefully tilt the box on itsside and hold it stationary while someone slides the contents out of the box.Ensure that anyone who operates the instrument has:• Received instructions in both general safety practices for laboratories andspecific safety practices for the instrument.• Read and understood all applicable Material Safety Data Sheets (MSDSs). See“About MSDSs” on page xviii.PHYSICAL INJURY HAZARD. Use this instrument asspecified by Applied Biosystems. Using this instrument in a manner not specified byApplied Biosystems may result in personal injury or damage to the instrument.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxvii

Safety and EMC Compliance InformationChemical SafetyChemical HazardWarningCHEMICAL HAZARD. Before handling any chemicals, refer tothe Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe allrelevant precautions.CHEMICAL HAZARD. All chemicals in the instrument, includingliquid in the lines, are potentially hazardous. Always determine what chemicals have beenused in the instrument before changing reagents or instrument components. Wearappropriate eyewear, protective clothing, and gloves when working on the instrument.CHEMICAL HAZARD. Four-liter reagent and waste bottles cancrack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safetycontainer with the cover fastened and the handles locked in the upright position. Wearappropriate eyewear, clothing, and gloves when handling reagent and waste bottles.CHEMICAL STORAGE HAZARD. Never collect or store wastein a glass container because of the risk of breaking or shattering. Reagent and wastebottles can crack and leak. Each waste bottle should be secured in a low-densitypolyethylene safety container with the cover fastened and the handles locked in theupright position. Wear appropriate eyewear, clothing, and gloves when handling reagentand waste bottles.About MSDSsObtainingMSDSsChemical manufacturers supply current Material Safety Data Sheets (MSDSs) withshipments of hazardous chemicals to new customers. They also provide MSDSs withthe first shipment of a hazardous chemical to a customer after an MSDS has beenupdated. MSDSs provide the safety information you need to store, handle, transport,and dispose of the chemicals safely.Each time you receive a new MSDS packaged with a hazardous chemical, be sure toreplace the appropriate MSDS in your files.You can obtain from Applied Biosystems the MSDS for any chemical supplied byApplied Biosystems. This service is free and available 24 hours a day.To obtain MSDSs:1. Go to https://docs.appliedbiosystems.com/msdssearch.html2. In the Search field, type in the chemical name, part number, or other informationthat appears in the MSDS of interest. Select a language, then click Search.3. Find the document of interest, right-click the document title, then select any ofthe following:• Open – To view the document• Print Target – To print the document• Save Target As – To download a PDF version of the document4. To have a copy of a document sent by fax or e-mail, select Fax or Email to theleft of the document title in the Search Results page, then click RETRIEVEDOCUMENTS at the end of the document list.5. After entering the required information, click View/Deliver SelectedDocuments Now.xviiiApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

Chemical Waste SafetyChemical SafetyGuidelines• Read and understand the Material Safety Data Sheets (MSDS) provided by thechemical manufacturer before you store, handle, or work with any chemicals orhazardous materials. (See “About MSDSs” on page xviii.)• Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing). For additional safety guidelines, consult the MSDS.• Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood). For additionalsafety guidelines, consult the MSDS.• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow themanufacturer’s cleanup procedures as recommended on the MSDS.• Comply with all local, state/provincial, or national laws and regulations relatedto chemical storage, handling, and disposal.Chemical Waste SafetyChemical WasteHazardHAZARDOUS WASTE. Refer to Material Safety Data Sheets andlocal regulations for handling and disposal.CHEMICAL WASTE HAZARD. Wastes produced by AppliedBiosystems instruments are potentially hazardous and can cause injury, illness, or death.CHEMICAL STORAGE HAZARD. Never collect or store wastein a glass container because of the risk of breaking or shattering. Reagent and wastebottles can crack and leak. Each waste bottle should be secured in a low-densitypolyethylene safety container with the cover fastened and the handles locked in theupright position. Wear appropriate eyewear, clothing, and gloves when handling reagentand waste bottles.Chemical WasteSafety Guidelines• Read and understand the Material Safety Data Sheets (MSDSs) provided by themanufacturers of the chemicals in the waste container before you store, handle,or dispose of chemical waste.• Provide primary and secondary waste containers. (A primary waste containerholds the immediate waste. A secondary container contains spills or leaks fromthe primary container. Both containers must be compatible with the wastematerial and meet federal, state, and local requirements for container storage.)• Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing). For additional safety guidelines, consult the MSDS.• Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).For additionalsafety guidelines, consult the MSDS.• Handle chemical wastes in a fume hood.• After emptying the waste container, seal it with the cap provided.• Dispose of the contents of the waste tray and waste bottle in accordance withgood laboratory practices and local, state/provincial, or national environmentaland health regulations.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxix

Safety and EMC Compliance InformationWaste ProfilesWaste DisposalA waste profile for the Applied Biosystems 7900HT Fast Real-Time PCR System isprovided in the Applied Biosystems 7900HT Fast Real-Time PCR System SitePreparation Guide.Waste profiles show the percentage compositions of the reagents in the waste streamgenerated during installation and during a typical user application, even though thetypical application may not be used in your laboratory.The waste profiles help you plan for the handling and disposal of waste generated byoperation of the instrument. Read the waste profiles and all applicable MSDSsbefore handling or disposing of chemical waste.If potentially hazardous waste is generated when you operate the instrument, youmust:• Characterize (by analysis if necessary) the waste generated by the particularapplications, reagents, and substrates used in your laboratory.• Ensure the health and safety of all personnel in your laboratory.• Ensure that the instrument waste is stored, transferred, transported, and disposedof according to all local, state/provincial, and/or national regulations.IMPORTANT! Radioactive or biohazardous materials may require special handling,and disposal limitations may apply.Electrical SafetyELECTRICAL SHOCK HAZARD. Severe electrical shock canresult from operating the Applied Biosystems 7900HT Fast Real-Time PCR Systemwithout its instrument panels in place. Do not remove instrument panels. Highvoltagecontacts are exposed when instrument panels are removed from theinstrument.FusesELECTRICAL SHOCK HAZARD. Improper fuses or highvoltagesupply can damage the instrument wiring system and cause a fire. Beforepowering on the instrument, verify that the fuses are properly installed and that theinstrument voltage matches the power supply in your laboratory.FIRE HAZARD. For continued protection against the risk offire, replace fuses only with fuses of the type and rating specified for the instrument.PowerELECTRICAL HAZARD. Grounding circuit continuity is vitalfor the safe operation of equipment. Never operate equipment with the groundingconductor disconnected.ELECTRICAL HAZARD. Use properly configured andapproved line cords for the voltage supply in your facility.ELECTRICAL HAZARD. Plug the system into a properlygrounded receptacle with adequate current capacity.xxApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

Physical Hazard SafetyOvervoltageRatingThe Applied Biosystems 7900HT Fast Real-Time PCR System has an installation(overvoltage) category of II, and is classified as portable equipmentPhysical Hazard SafetyMoving PartsPHYSICAL INJURY HAZARD. Moving parts can crush andcut. Keep hands clear of moving parts while operating the instrument. Disconnectpower before servicing the instrument.PHYSICAL INJURY HAZARD. Do not operate the instrumentwithout the arm shield in place. Keep hands out of the deck area when the instrumentis spotting.Biological Hazard SafetyGeneralBiohazardBIOHAZARD. Biological samples such as tissues, body fluids,and blood of humans and other animals have the potential to transmit infectiousdiseases. Follow all applicable local, state/provincial, and/or national regulations.Wear appropriate protective eyewear, clothing, and gloves. Read and follow theguidelines in these publications:• U.S. Department of Health and Human Services guidelines published inBiosafety in Microbiological and Biomedical Laboratories (stock no. 017-040-00547-4; http://bmbl.od.nih.gov)• Occupational Safety and Health Standards, Bloodborne Pathogens(29 CFR§1910.1030; http://www.access.gpo.gov/nara/cfr/waisidx_01/29cfr1910a_01.html).Additional information about biohazard guidelines is available at:http://www.cdc.govLaser SafetyLaserClassificationThe Applied Biosystems 7900HT Fast Real-Time PCR System uses a Class 3B laser.Under normal operating conditions, the instrument laser is categorized as a Class 3Blaser. When safety interlocks are disabled during certain servicing procedures, thelaser can cause permanent eye damage, and, therefore, is classified under thoseconditions as a Class 3B laser.The Applied Biosystems 7900HT Fast Real-Time PCR System complies with 21CFR, 1040.10 and 1040.11, as applicable.The Applied Biosystems 7900HT Fast Real-Time PCR System has been tested to andcomplies with the “Radiation Control for Health and Safety Act of 1968 PerformanceStandard CFR 1040.”Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxxi

Safety and EMC Compliance InformationThe Applied Biosystems 7900HT Fast Real-Time PCR System has been tested to andcomplies with standard EN60825-1, “Radiation Safety of Laser Products, EquipmentClassification, Requirements, and User’s Guide.”Laser SafetyRequirementsAdditional LaserSafetyInformationTo ensure safe laser operation:• The system must be installed and maintained by an Applied BiosystemsTechnical Representative.• All instrument panels must be in place on the instrument while the instrument isoperating. When all panels are installed, there is no detectable radiation present.If any panel is removed when the laser is operating (during service with safetyinterlocks disabled), you may be exposed to laser emissions in excess of theClass 3B rating.• Do not remove safety labels or disable safety interlocks.Refer to the user documentation provided with the laser for additional information ongovernment and industry safety regulations.LASER HAZARD. Lasers can burn the retina causingpermanent blind spots. Never look directly into the laser beam. Remove jewelry andother items that can reflect the beam into your eyes. Do not remove the instrumenttop or front panels. Wear proper eye protection and post a laser warning sign at theentrance to the laboratory if the top or front panels are removed for service.LASER BURN HAZARD. An overheated laser can causesevere burns if it comes in contact with the skin. DO NOT operate the laser when itcannot be cooled by its cooling fan. Always wear appropriate laser safety goggles.Bar Code Scanner Laser SafetyLaserClassificationLaser SafetyRequirementsThe bar code scanner included with the Applied Biosystems 7900HT Fast Real-TimePCR System is categorized as a Class 2 (II) laser.Class 2 (II) lasers are low-power, visible-light lasers that can damage the eyes.Never look directly into the laser beam. The scanner is designed to prevent humanaccess to harmful levels of laser light during normal operation, user maintenance, orduring prescribed service operations.LASER HAZARD. Class 2 (II) lasers can cause damage to eyes.Avoid looking into a Class 2 (II) laser beam or pointing a Class 2 (II) laser beam intoanother person’s eyes.xxiiApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

Workstation SafetyWorkstation SafetyCorrect ergonomic configuration of your workstation can reduce or prevent effectssuch as fatigue, pain, and strain. Minimize or eliminate these effects by configuringyour workstation to promote neutral or relaxed working positions.MUSCULOSKELETAL AND REPETITIVE MOTIONHAZARD. These hazards are caused by potential risk factors that include but are notlimited to repetitive motion, awkward posture, forceful exertion, holding staticunhealthy positions, contact pressure, and other workstation environmental factors.To minimize musculoskeletal and repetitive motion risks:• Use equipment that comfortably supports you in neutral working positions andallows adequate accessibility to the keyboard, monitor, and mouse.• Position the keyboard, mouse, and monitor to promote relaxed body and headpostures.Safety and Electromagnetic Compatibility (EMC) StandardsThis section provides information on:• U.S. and Canadian Safety Standards• Canadian EMC Standard• European Safety and EMC Standards• Australian EMC StandardsU.S. andCanadian SafetyStandardsCanadian EMCStandardEuropean Safetyand EMCStandardsThis instrument has been tested to and complies with standard UL 3101-1, “SafetyRequirements for Electrical Equipment for Laboratory Use, Part 1: GeneralRequirements.”This instrument has been tested to and complies with standard CSA 1010.1, “SafetyRequirements for Electrical Equipment for Measurement, Control, and LaboratoryUse, Part 1: General Requirements.”This instrument has been tested to and complies with ICES-001, Issue 3: Industrial,Scientific, and Medical Radio Frequency Generators.SafetyThis instrument meets European requirements for safety (Low Voltage Directive73/23/EEC). This instrument has been tested to and complies with standardsEN 61010-1:2001, “Safety Requirements for Electrical Equipment for Measurement,Control and Laboratory Use, Part 1: General Requirements” and EN 61010-2-010,“Particular Requirements for Laboratory Equipment for the Heating of Materials.”EMCThis instrument meets European requirements for emission and immunity (EMCDirective 89/336/EEC). This instrument has been tested to and complies withstandard EN 61326 (Group 1, Class B), “Electrical Equipment for Measurement,Control and Laboratory Use – EMC Requirements.”Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fmxxiii

Safety and EMC Compliance InformationAustralian EMCStandardsThis instrument has been tested to and complies with standard AS/NZS 2064,“Limits and Methods Measurement of Electromagnetic Disturbance Characteristicsof Industrial, Scientific, and Medical (ISM) Radio-frequency Equipment.”xxivApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Safety.fm

Product Overview 11In This Chapter System Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2Section 1.1 Getting to Know the Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-37900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9Instrument Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10Section 1.2 Getting to Know the Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15Managing Sequence Detection System Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17Section 1.3 SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-21About the SDS Enterprise Database Feature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22About the SDS Enterprise Database Software Suite. . . . . . . . . . . . . . . . . . . . . . . 1-25Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-1DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

EnterV C X Z < M N B > ? / F D S A K J H G L " 'R E W Q I U Y T O P [ ]- = Backspace9 0` _5 6 7 81 2 3 4+~Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 1 F F1203 2 1Enter4 6 58 7 9locklock lockl scro capsnumDownPageUpPageEndDeleteInsert Home*+Lock CapsTabShiftCtrl AltAlt CtrlShiftSysRqen ScrPrintBreakLockl Scro PauseNum LockChapter 1Product OverviewSystem OverviewAbout the7900HTInstrumentThe Applied Biosystems 7900HT Fast Real-Time PCR System is asecond-generation sequence detection system instrument designed for automated,high-throughput detection of fluorescent PCR-related chemistries. The instrument iscapable of real-time, end-point, and dissociation curve analysis of assays arrayed onmultiple formats. The 7900HT instrument is optimized for use withApplied Biosystems chemistries for nucleic acid quantification and detection.Figure 1-1 The Applied Biosystems 7900HT Fast Real-Time PCR System withthe Automation AccessorySupported Runsand ChemistriesThe Applied Biosystems 7900HT Fast Real-Time PCR System provides two types ofruns to support the variety of PCR-related chemistries available fromApplied Biosystems and affiliated companies.End-Point (Plate Read) ChemistryIn an end-point run, the thermal cycling of prepared plates containing reagents andtemplate is performed on a dedicated thermal cycler. Following the PCR, the 7900HTinstrument is then used to collect a plate-read or a single reading of the fluorescenceresulting from the completed reactions. Allelic Discrimination is an example of anend-point chemistry that is directly supported by the SDS software.Real-Time ChemistryIn a real-time run, the thermal cycling of prepared plates containing reagents andtemplate is performed on the 7900HT instrument. During the run, the instrumentcollects data at each cycle of the PCR. The resulting data provides a chronologicalseries of measurements of the fluorescence resulting from the reactions. Examples ofreal-time chemistry include absolute quantification, relative quantification anddissociation curve analysis.1-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

7900HT FAST Real-Time PCR SystemSection 1.1 Getting to Know the HardwareSection 1.1Getting to Know the HardwareIn This Section 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4Computer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6Bar Code Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7Zymark Twister Microplate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9Instrument Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10InstrumentComponentsThe Applied Biosystems 7900HT Fast Real-Time PCR System consists of thecomponents shown in Figure 1-2.7900HT Instrument(see page 1-4)Microsoft ® Windows ®Compatible Computer(see page 1-6)GR20097900HTFront view with RobotGR2009Fixed-PositionBar Code Reader(see page 1-7)Zymark ® TwisterMicroplate Handler(see page 1-8)ExtendedCapacity Stacks(see page 1-7)Hand-Held BarCode Reader(see page 1-7)Figure 1-2SystemComponents of the Applied Biosystems 7900HT Fast Real-Time PCRApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-3DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product Overview7900HT InstrumentInternalComponentsThe 7900HT instrument contains the hardware used for thermal cycling anddetection of fluorescent chemistries (see page 1-2). Figure 1-3 illustrates the majorsubcomponents of the instrument described in detail below.Do not remove the cover to the Applied Biosystems 7900HT FastReal-Time PCR System. Only a qualified Applied Biosystems service engineer mayrepair or adjust the internal components of the 7900HT instrument. Failure to complycan result in serious injury and/or damage to the instrument.LaserCCDcameraReaderOpticssystemGR2112TrayDoorHeated clampAutomated platehandling systemSample block moduleFigure 1-3Internal Components of the 7900HT InstrumentAutomated PlateHandling SystemHeated ClampPHYSICAL HAZARD. Keep hands and loose clothing awayfrom the instrument tray and door at all times during instrument operation. The7900HT instrument contains several internal components that can cause seriousphysical injury.The 7900HT instrument features an automated plate-handling system to provide easyloading and removal of plates from the instrument. In combination with theautomation module, the plate-handling system allows unattended operation of theinstrument.PHYSICAL INJURY HAZARD. Hot Surface. Use care whenworking around this area to avoid being burned by hot components.The 7900HT instrument has a heated clamp that provides optimal heat transfer anduniform heating during thermal cycling. When the instrument tray loads a plate, theclamp applies a downward pressure of 70 lbs (31.8 kg) onto the consumable. Duringthe run, the clamp maintains a constant temperature of 105 °C (±3 °C) to preventcondensation within the consumable wells.1-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

7900HT InstrumentInterchangeableThermal CyclerBlocksThe 7900HT instrument features a Peltier-based, interchangeable sample blockmodule based on the technology established in the GeneAmp ® PCR System 9700thermal cycler. The sample block module houses an internal Peltier heating/coolingunit (Figure 1-4). The sample block module is made of aluminum to provide anoptimal thermal transfer rate between the block and the optical plate or theTaqMan ® Low Density Array.PHYSICAL INJURY HAZARD. Hot Surface. Use care whenworking around this area to avoid being burned by hot components.Connection to the instrument(Do Not Touch)WarningGR2027Circuitry(Do Not Touch)GR2028GR2028Block insideinstrumentSample block(Do Not Touch)Top viewHeat sinks(Do Not Touch)Bottom viewFigure 1-4Components of the Interchangeable Thermal Cycler BlockThe interchangeable sample block module provides:• Wide temperature range: 4–99.9 °C• Accuracy: ±0.25 °C from 35–99.9 °C• Heat/cool rate: 1.5 °C per second• Temperature uniformity: ±0.5 °C (measured 30 sec after the clock starts)• Long-term stability and high reliability• Support for multiple consumable formats (see page 1-9)• Several different modes of operation including 9600 mode and programmabletemperature ramps (see page 3-21)• Reduced instrument downtime by allowing immediate replacement of the block(see page 7-6)• Easy access to the sample block for troubleshooting and maintenance(see page 7-6)Optics SystemIMPORTANT! Do not remove the cover to the 7900HT instrument. Only a qualifiedApplied Biosystems service engineer may repair or adjust the internal components.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-5DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewComputerFunctionsA Microsoft Windows-compatible computer is required to operate the AppliedBiosystems 7900HT Fast Real-Time PCR System and the Zymark TwisterMicroplate Handler. Through the SDS and Automation Controller Software, thecomputer:• Provides the interface for programming and operating the instrument• Coordinates the operation of the 7900HT instrument, automation module, andthe bar code readers• Provides storage and management of the data produced by the 7900HTinstrument• Provides the local area network (LAN) connection to the SDS EnterpriseDatabase (if available)SystemRequirementsThe system requirements for the computer used to operate the 7900HT instrumentvary with the version of the SDS software. To determine the system requirements foryour instrument, check the release notes accompanying your version of the SDSsoftware.The release notes for the SDS software are in the following location:D:\AppliedBiosystems\SDS2.2.1\ReleaseNotes.txtNote: You can use the Microsoft Notepad, Wordpad, or Word software to read therelease notes text file.Hard DrivePartitionsDuring installation of the 7900HT instrument, the Applied Biosystems service engineerpartitions the computer hard drive to create the logical drives shown in the table below.Primary Hard DriveDriveSize(GB)ContainsC 2 Operating system files **Applied Biosystems recommends that you do not install programs to the C drive. Thecomputer will boot faster if the C drive contains only the operating system.†Software required for the operation of the bar code reader.D ≥25 • SDS Software• Automation Controller Software• Zymark Twister Software• LAVA Software †• Additional Third-Party Software• SDS 7900HT Documents• (Optional) SDS EnterpriseDatabase Client SoftwareNote: The partitions shown above represent the basic drive architecture required bythe SDS software. The actual number and size of the partitions and drives availableon your computer vary depending on the model of computer accompanying yourinstrument.1-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

7900HT FAST Real-Time PCR SystemBar Code ReadersBar Code ReadersDescriptionThe Applied Biosystems 7900HT Fast Real-Time PCR System includes two bar codereaders for data entry and plate recognition:• A hand-held bar code reader for scanning plates manually• A fixed-position bar code reader for automatically scanning plates as they areloaded into the instrument (available only with the Automation Accessory)Both bar code readers use a 670 nm Class II laser to scan plates and are capable ofreading Code 128 (alphanumeric), which supports 128 ASCII character bar codes.Locations of theBar CodeReadersGR20097900HTFront view with RobotGR2009Fixed-Position Bar Code ReaderHand-Held Bar Code ReaderSplitterTo computerkeyboard portGR2018~~~To keyboard(Shown with cover removed)GR2015Figure 1-5Locations of the Bar Code Readers of the 7900HT InstrumentUsing theBar CodeReadersLASER HAZARD. Exposure to direct or reflected laser lightcan burn the retina and leave permanent blind spots. Never look into the laser beam.Remove jewelry and anything else that can reflect the beam into your eyes. Protectothers from exposure to the beam.For more information on the bar code readers, see:Using the Hand-Held Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-27Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-7DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewZymark Twister Microplate HandlerDescriptionThe Zymark ® Twister Microplate Handler coordinates plate handling for the 7900HTinstrument, permitting 24-hour unattended operation. The arm provides a 310-degreerotational swing that permits access to the 7900HT instrument drawer, up to fiveplate stacking areas, and the fixed-position bar code reader.Zymark TwisterMicroplateHandlerComponentsZymark TwisterMicroplate Handler(cross-sectional view of the gripper)Plate-sensorswitchGripperAdjustmentknobFixed-positionbar code readerExpansionstacksPlate stackFigure 1-6Components of the Zymark Twister Microplate HandlerPlate PositionsX4312Zymark Twister Microplate Handler(top view)Bar codeWell A1Figure 1-7Plate Positions of the Zymark Twister Microplate HandlerUsing theZymark TwisterMicroplateHandlerLASER HAZARD. Exposure to direct or reflected laser lightcan burn the retina and leave permanent blind spots. Never look into the laser beam.Remove jewelry and anything else that can reflect the beam into your eyes. Protectothers from exposure to the beam.For information on the Zymark Twister Microplate Handler, see:Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4Starting and Configuring the Automation Controller Software for Operation . . .4-39Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-351-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Compatible ConsumablesCompatible ConsumablesConsumables forUse with the7900HTInstrumentThe interchangeable sample block modules of the 7900HT instrument allow it tosupport a variety of consumable formats listed in Table 1-1.Table 1-1Compatible ConsumablesConsumableABI PRISM 384-Well OpticalReaction PlatesABI PRISM 96-Well OpticalReaction PlatesOptical 96-Well Fast ThermalCycling PlatesTaqMan ® Low Density ArraysCompatible Seals• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)• Compression Pads:– Standard pad for manual operation– ABI PRISM Snap-On Compression Pad(reusable)• 96-Well Plate Seals:– ABI PRISM Optical Adhesive Covers(quantity 100)– Optical Adhesive Covers (quantity 25)– ABI PRISM Optical Caps (flat caps only)• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)None (self sealing)CompressionPads for theABI PRISM96-Well OpticalReaction PlatesConsumableRequirementsTo ensure optimal heat transfer, Applied Biosystems recommends using compressionpads when running the ABI PRISM 96-Well Optical Reaction Plates.If you are going to run the plate:• Individually using the SDS software, then apply a standard compression pad• As part of a batch using the Automation Controller Software, then apply anABI PRISM Snap-On Compression PadIMPORTANT! The ABI PRISM Snap-On Compression Pad/plate assembly may stillbe hot after the Zymark ® Twister Microplate Handler loads it into the Plate Handler’soutput stack. Please wait at least 30 seconds before manually handling the Snap-OnCompression Pad/plate assembly.IMPORTANT! Performance of the Optical 96-Well Fast Thermal Cycling Plates withthe Zymark Twister Microplate Handler has not been verified. Therefore, whenrunning multiple Optical 96-Well Fast Plates on the 7900HT instrument, AppliedBiosystems recommends that you load and unload the plates manually.See Appendix C, “Kits, Reagents, and Consumables,” for a list of availableconsumables, requirements, and purchasing instructions.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-9DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

HI-POTA B C DChapter 1Product OverviewInstrument ConnectionsElectricalConnectionsFigure 1-8 and Table 1-2 illustrate the electrical connections of the AppliedBiosystems 7900HT Fast Real-Time PCR System components.PowerAHPowerPowerHI-POTA B C DGR2020BCEADFGPowerEGPower CCommunications CablePower CableFigure 1-8SystemConnections of the Applied Biosystems 7900HT Fast Real-Time PCRTable 1-2SystemConnections of the Applied Biosystems 7900HT Fast Real-Time PCRCable Type Connects… To…A Communication Computer (Monitor Port) MonitorB Comm/Power Computer (Mouse Port) Mouse (not shown)C Serial Computer (Serial Port 1) 7900HT InstrumentD Comm/Power Computer (Keyboard Port) Hand-held Bar Code ReaderE Communication Computer (Serial Port 2) Plate Handler (Port C)F Ethernet Network Computer (Ethernet Port)G *Comm/Power Computer (ISA Card 1) Fixed-Position Bar Code ReaderH Comm/Power Bar Code Reader Cable Keyboard (not shown)*See Figure 1-9 on page 1-11.1-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Instrument ConnectionsFixed-PositionBar Code ReaderBar CodeReader CableTo Lava Card(ISA Card 1)Power supplyPowerGFigure 1-9Fixed-Position Bar Code Reader ConnectionUniversal VoltageAccessory Kit(P/N 4334482)IEC 320 4-PositionUniversal Power Strip(PN 4333969)IEC 320 North America × (1)IEC 320 ContinentalUniversal Jumper Cord set × (4)Jumper Cord set for Japan × (4)(see below for use in Japan)IEC 320 U.K./Ireland × (1)IEC 320 Australia/New Zealand × (1)IEC 320 Japan × (1)In Bag Marked “JAPAN ONLY”Figure 1-10To computer, monitor, hand-held barcode reader, and the Plate HandlerNote: The order is not critical.Universal Power StripNorth America: 250V@15A MAXEurope and all other locations: 250V@10A MAXTo install the Universal Voltage Accessory Kit (All Countries Except Japan):1. Connect one universal jumper to each accessory (see below for use in Japan).2. Connect the other end of the power jumpers to the power strip outputs.3. Choose the correct country specific power input cord for the geographical regionand connect it to the power strip input.4. Power off all instrument accessories (monitor, computer, and Plate Handler).5. Connect the power-input cord to the AC outlet.6. Power on accessories.7. Discard unused cord sets.Usage Guidelines for Japan1. Use only the cord sets supplied in the bag marked “JAPAN ONLY.”2. Discard all other unused cord sets.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-11DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product Overview1-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Section 1.2 Getting to Know the SoftwareSection 1.2Getting to Know the SoftwareIn This Section Software Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14SDS Software Related Files and Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15Managing Sequence Detection System Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-17Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-13DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewSoftware ComponentsComponents ofthe SDS SoftwareSuiteThe Applied Biosystems 7900HT Fast Real-Time PCR System uses a suite ofapplications to set up, run, and analyze experiments completed on the 7900HTinstrument.Table 1-3Standard SDS SoftwareSoftwareFunctionSDS Software • Constructs and edits plate document files• Performs initial and end analysis of raw data fromallelic discrimination, relative quantification, andabsolute quantification runs• Saves, prints, and exports run dataJava Runtime EnvironmentAdditional files and software used to run the SDSsoftwareIMPORTANT! Do not delete the Java RuntimeEnvironment files. These files are crucial to the operationof the SDS software.7900HT Instrument Firmware • Controls the most basic operations of the 7900HTinstrument• Controlled by commands sent from the computer• Links the commands issued by the SDS software andthe operation of the 7900HT instrumentTable 1-4Software for Automated or SDS Enterprise Database OperationSoftwareAutomation ControllerSoftwareZymark Twister SoftwareLAVA SoftwareSDS Enterprise DatabaseClient SoftwareFunction• Controls and coordinates the action of the 7900HTinstrument and the automation module• Initiates and controls the sequence detection run• Acquires data during the runUsed to calibrate the Zymark Twister Microplate Handler.Used to align the fixed-position bar code reader.Links the SDS software to an SDS Enterprise Databaseon the network.Note: See page 1-21 for more information on thesoftware components of the SDS Enterprise Database.1-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

SDS Software Related Files and FormatsSDS Software Related Files and FormatsFiles Used andCreated by theSDS SoftwareIMPORTANT! Never move or delete program files or directories for SDS-relatedsoftware unless specifically directed to do so by an Applied Biosystemsrepresentative or documentation.The SDS software uses many different files and directories (Table 1-5). Some ofthese files store run data and calibration data: others are required to run the 7900HTinstrument.Table 1-5Common SDS FilesFile Type Ext. Icon Description/CommentsPlate Document FilesSDS 7900HTDocumentSDS 7900HTMultiple PlateDocumentSDS 7900HTTemplateDocument **.sds • Serves as a virtual representation of a singleconsumable used to contain samples andreagents during a sequence detection run• Contains all sample and assay data (names,locations, reporter dye) contained by theconsumable• Contains instructions for running the instrument(thermal cycling and data collection parameters)• Stores data gathered during the run*.sdm • Used to analyze relative quantification data(small studies)*.sdt • Used to create plate documents• Can store assay data, thermal cycling, and datacollection parametersAssay PlateDocumentSetup Files*.txtImported/Exported FilesUsed to create plate documentsExported DataFiles*.txt • Contains exported raw or analyzed data for all ora select group of wells on a plate document• Uses (*.txt) a tab-delimited format (compatiblewith most spreadsheet applications)JPEG † GraphicFile*.jpg ‡• Most panes and plots of the plate document canbe exported as JPEG graphic files• Compatible with most word processing andspreadsheet applications• Can be incorporated directly into HTML or XMLdocuments*The use of plate document templates is optional but useful as a timesaving device forexperiments where samples are run on plates with identical assay configurations.†Joint Photographic Experts Group‡Icon will varies depending on the application associated with the image file typeApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-15DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewPlate DocumentFile SizeThe SDS software can produce plate document files of varying sizes, depending onthe type of runs for which they are created. Table 1-6 lists the average sizes of typicalfiles produced by the SDS software.Table 1-6Average Plate Document File SizesRun TypeApplicationUncompressedFile Size *CompressedPlate-Read Allelic Discrimination 150 to 180 KB 70 to 90 KBReal-Time †Absolute QuantificationRelative Quantification15 to 25 MB ‡10 to 15 MB*Compressed file sizes are estimates based on standard compression using the WinZip ®utility. For more information, see “Archiving SDS Files” on page 7-54.†The maximum file sizes displayed above are nominal for real-time runs (absolute or relativequantification). File size can increase depending on the plate document’s data collectionoptions.‡The file size for a real-time run depends on the length of the run and the configuration of thedata collection options in the plate document. Longer runs and runs configured to collectdata at multiple stages of the method can be considerably larger than the 15 to 25 MBaverage.IMPORTANT! Because of memory constraints, the SDS Enterprise Database cannotsave real-time plate documents greater than 40 MB.1-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

7900HT FAST Real-Time PCR SystemManaging Sequence Detection System DataManaging Sequence Detection System DataSystemOperation andDataflowStandard Modesof OperationData management strategy is a crucial element of successfully integrating theApplied Biosystems 7900HT Fast Real-Time PCR System into a laboratoryworkflow. During a single 24-hour period of real-time operation, the 7900HTinstrument can produce more than 200 MB of data. To successfully manage theinformation produced by the 7900HT instrument, you should have a basicunderstanding of how data is collected, stored, and processed throughout theoperation of the instrument.The Applied Biosystems 7900HT Fast Real-Time PCR System has three standardmodes of operation depending on the accessories purchased with the base instrument• Stand-Alone Operation (see below)• Automated Operation (see page 1-18)• Database Operation (see page 1-18)Stand-Alone OperationThis mode uses the most basic configuration of the Applied Biosystems 7900HT FastReal-Time PCR System (Figure 1-11) where the 7900HT instrument is used without anyadditional components. In this configuration, a technician runs plates individually usingthe SDS software which stores all data on the computer hard drives.ComputerGR21797900HT raveninstrument frontSDS SoftwareApplied Biosystems 7900HTFast Real-Time PCR SystemInstrument Firmware12Plate DocumentCreatedPlate Document Usedto Run a PlateHard drive(s)3Thermal Cycling andSequence Detection5Raw Data Saved tothe Plate Document*.sdsfile*.sdtfile4Data CollectionSerialCable6Plate DocumentOpened and AnalyzedFigure 1-11Stand-Alone Operation of the 7900HT InstrumentApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-17DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

7900HT FAST Real-Time PCR SystemChapter 1Product OverviewAutomated OperationIn this mode, the Applied Biosystems 7900HT Fast Real-Time PCR System uses anAutomation Accessory (Zymark Twister Microplate Handler and fixed-position barcode reader), to provide high-throughput operation suitable for small- tomedium-scale studies. With this configuration (Figure 1-12), a technician can runbatches of plates unattended using the Automation Controller Software. As inStand-Alone operation, the computer stores all data for operation of the instrumentand provides the software for analyzing the run data.ComputerSDS SoftwareAutomation ControllerSoftware1Plate DocumentsCreatedGR2009GR20097900HTFront view with RobotApplied Biosystems 7900HTFast Real-Time PCR SystemInstrument Firmware3Thermal Cycling andSequence Detection25Plate document filesadded to and run fromthe plate queueRaw Data Saved tothe Plate DocumentsHard drive(s)*.sdsfiles*.sdtfile4Data CollectionSerialCable6Plate DocumentOpened and AnalyzedFigure 1-12Automated Operation of the of the 7900HT InstrumentDatabase OperationFor this mode, the Applied Biosystems 7900HT Fast Real-Time PCR System usesthe SDS Enterprise Database and the Automation Accessory, to provide maximumhigh-throughput potential. In this configuration (Figure 1-13 on page 1-19), the7900HT instrument has the storage capacity and analysis capability to supportmedium- to large-scale gene expression and genotyping studies. Moreover, the SDSEnterprise Database functions as a repository for all SDS-related data produced bythe networked client computers used for data collection and analysis.1-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F 1 F12~ _` 1 2 3 46 7 8 9 0 - = BackspaceQ W E R T Y U I O P [ ]A S D FH" G J K L 'X C VN MTabCaps LockShiftCtrl AltPrintScr enSysRq2 3 Enter15 64Scro lLockPauseBreakInsert HomeDelete EndShiftAlt Ctrllock lock lockscro lcapsnum*num caps scro llock lock lockPageUpPageDownNum Lock*7 8 94 5 61F10F9+7 9 8V C X Z < M N B > ? /EnterF D S A K J H G L " 'P [ ]O T Y U IQ R- = Backspace9 0` _5 6 7 81 2 3 4+~+2 30EnterEsc0Num LockDownPageUpPageBreakLockl Scro PauseEndDeleteInsert HomeAlt CtrlShiftSysRqen ScrPrintShiftCtrl AltLock CapsTabManaging Sequence Detection System DataApplied Biosystems 7900HTFast Real-Time PCR SystemInstrument Firmware3Thermal Cycling andSequence DetectionData Collection Client(Desktop Computer)Automation ControllerSoftware2Plate documents runfrom the databaseF 1 F12F7 F8F5 F6E WF3 F4F1 F24 5SerialData CollectionCableRaw and Analyzed DataSaved to the DatabaseDatabase ServerSDS EnterpriseDatabaseEthernetPlateDocumentsAnalysisSessionsStudiesAnalysis Client (Desktop Computer)Instrument Firmware6Plate DocumentOpened and Analyzed1EthernetPlate DocumentsCreated5+Enter< > ? Z B /RQ Manager SoftwareHard drive(s)6Analysis SessionsAnalyzed as StudiesSNP Manager Software6Analysis SessionsAnalyzed as StudiesAssay PlateSetup FilesFigure 1-13 Automated Operation of the of the 7900HT Instrument withSDS Enterprise DatabaseApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-19DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product Overview1-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Section 1.3 SDS Enterprise DatabaseSection 1.3SDS Enterprise DatabaseIn This Section About the SDS Enterprise Database Feature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-22About the SDS Enterprise Database Software Suite. . . . . . . . . . . . . . . . . . . . . . . 1-25Database Design and Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . 1-27Supporting API Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-28Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-21DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewAbout the SDS Enterprise Database FeatureAbout theDatabaseAccess ControlNetworkArchitectureThe SDS software can interface directly with an Applied Biosystems SDS EnterpriseDatabase as part of a laboratory information management system (LIMS). The SDSEnterprise Database is a relational, Oracle-based repository designed specifically tostore SDS data produced by an Applied Biosystems 7900HT Fast Real-Time PCRSystem. In addition to serving as a common repository for SDS data, the databaseprovides user authentication, robust and scalable data management, and flexiblearchive capabilities. The database also provides the infrastructure and tools necessaryto integrate a 7900HT instrument into a laboratory workflow made up of existingdata management solutions.The SDS Enterprise Database provides a multi-level user security system thatrestricts access to the database and to most functions of the SDS software and7900HT instrument. The database provides the SDS User Account Manager softwarewith the client version of the SDS software for managing user accounts. The UserManager allows users with Administrator privileges to assign privileges andpasswords for user accounts, and to add and remove them. This guide indicates theaccess privileges required to perform each task using a short note above eachprocedure.Note: See the SDS Enterprise Database for the Applied Biosystems 7900HT FastReal-Time PCR System Administrators Guide (PN 4351669) for a completedescription of user accounts for the SDS Enterprise Database.The SDS Enterprise Database is a relational, Oracle-based repository designed to storeSDS data produced by Applied Biosystems 7900HT Fast Real-Time PCR Systeminstruments. In addition to serving as a common repository for SDS data, the databaseprovides user authentication, robust and scalable data management, and flexible archivecapabilities. The database also provides the infrastructure and tools necessary tointegrate a 7900HT instrument into a laboratory workflow made up of existing datamanagement solutions.The SDS Enterprise Database supports a basic two-tier, server-client architecturewithin an Ethernet (10Base-T) or Fast Ethernet (100Base-T) network environment.The database can function as part of an isolated star network via the SMC routerprovided with the database (Figure 1-14), or as a node on a larger bus network(Figure 1-15). The server computer uses a static IP address to simultaneously serveup to 5 clients configured for either DHCP or static TCP/IP operation.1-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

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About the SDS Enterprise Database Software SuiteAbout the SDS Enterprise Database Software SuiteDatabaseManagementSoftwareThe SDS Enterprise Database provides software utilities to help integrate thedatabase into laboratory workflows as describes in:Database Modules for Large-Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-25Database Management Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-26Database Modules for Large-Scale AnalysisAbout theAnalysis ModulesApplied Biosystems offers two analysis modules (the SNP Manager Software andRQ Manager Software) for conducting medium- to large-scale analysis ofSNP/genotyping and gene expression on the SDS Enterprise Database (Figure 1-16).SDS EnterpriseDatabaseStudies/Run DataAnalysis SessionsSNP ManagerSoftwareSNP/GenotypingStudyRQ ManagerSoftwareRelativeQuantification StudyFigure 1-16SDS Enterprise Database Applications for Large-Scale AnalysisAbout theSNP ManagerSoftwareAbout theRQ ManagerSoftwareThe SNP Manager Software is a stand-alone application used to perform medium- tolarge-scale analyses of SNP (genotyping) data stored on the database. The softwarecan make comparative analyses of up to 5,000 plates or approximately 1.9 million5´ nuclease reactions run on an Applied Biosystems 7900HT Fast Real-Time PCRSystem. See the SNP Manager Software User Guide (PN 4351671) for moreinformation.The RQ Manager Software is a stand-alone application used to perform medium- tolarge-scale analyses of relative quantification (gene expression) data stored on anSDS Enterprise Database. The software can make comparative analyses of over97 detectors (96 target genes and 1 endogenous control) across 200 plates, orapproximately 153,600 multiplexed reactions run on an Applied Biosystems 7900HTFast Real-Time PCR System. See the RQ Manager Software User Guide(PN 4351670) for more information.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-25DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewDatabase Management UtilitiesFunctionalSummaryThe SDS Enterprise Database includes a suite of software applications thatadminister the use and content of the database. Figure 1-17 summarizes the basicfunction of each tool and the type of data it handles.PlateDocumentsSDSDocumentLoaderSDSTemplateLoaderPlateDocumentTemplatesSDSDocumentCreatorPlateDocumentsSDS EnterpriseDatabaseSDS UserAccountManagerSDSDocumentExtractorSDSDatabaseArchiverPlateDocumentsArchiveFiles (.arc)Figure 1-17Functional Summary of the Database Management UtilitiesSDS DocumentCommand LineToolsSDS DatabaseArchiverThe SDS Enterprise Database Software Suite includes four command-line utilitiesfor managing plate documents, plate document templates, and run data produced bythe 7900HT instrument.• SDS Document Creator – Creates plate documents in the database using thecontents of a plate document template and an assay plate setup file• SDS Document Loader – Populates the database with the contents of existing platedocuments• SDS Document Extractor – Creates plate documents from the data contained in thedatabase• SDS Template Loader – Populates the database with the contents of existing platedocument templatesFor detailed information on these utilities, see the SDS Enterprise Database for theApplied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide(PN 4351669).The SDS Archiver Software installs with the version of the SDS software thatincludes the database client software. The software allows users with Administratorprivileges to download session (run) data from the database, create archive files fromthem, and restore archived data to the database. See the SDS Enterprise Database forthe Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide(PN 4351669) for more information.1-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F 1 F12~ _ +` 1 2 3 4 5 6 7 8 9 0 - = BackspaceQ W E R T Y U I O P [ ]A S D FH" G J K L ' ?/EnterThe RQ Manager, SNPManager, or SDS softwareis used to download andanalyze analysis sessionsor studies.DesktopComputer• Studies• Run DataDatabaseServer7900HTInstrumentGR21797900HT raveninstrument front• Run Data• AnalysisSessions3The software runs each plateusing the method stored inthe downloaded platedocument. During the run,the software saves the rawdata to the plate document inthe database.4Immediately after each run, the softwareperforms a primary analysis(multicomponenting and normalization)of the raw data. The software then savesthe analyzed data to the database as ananalysis session and attaches it to theappropriate study.Figure 1-18SDS Enterprise Database Data Flow DiagramApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-27DRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Chapter 1Product OverviewTypes ofRetrievable DataPersisted Plate DocumentsPersisted plate documents include the equivalent information stored in SDS platedocuments. Plate documents have a 1:1 correspondence with the optical plate or LowDensity Array they represent. Each persisted plate documents consists of a collectionof plate setup information for running a plate on the 7900HT instrument including:• Sample/well assignments,• Marker/detector definitions and well assignments,• Marker/detector tasks and quantity,• Plate bar codeAnalysis SessionsAn analysis session is a set of analysis results derived from a single raw data setassociated with a single plate document analyzed by the SDS software. Multipleanalysis sessions can be associated with the same plate document. Each analysissession has a unique name used to distinguish it from the other sessions for the sameplate document.Session data exists in two states on the database: attached or unattached. Attachedsessions are those that have been assigned to a specific study. Session data can beattached to a study by the Automation Controller Software or by using the RQManager or SNP Manager Software.IMPORTANT! After you attach a session to a study, you cannot add it to anotherstudy until you unattach it. See the RQ Manager Software User Guide or SNPManager Software User Guide for instructions on removing attached sessions from astudy.RQ and SNP StudiesThe SDS Enterprise Database software uses a system of studies to organize thesecondary analysis of session data produced by an Applied Biosystems 7900HT FastReal-Time PCR System. The central purpose of using the study system is to groupthe analysis session data for analysis using the SNP Manager or the RQ ManagerAnalysis Software.Supporting API DocumentationTechnologySupportThe SDS Enterprise Database includes a copy of the SDS Enterprise Database forthe Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide,which provides information for integrating the database into a laboratory workflow.The guide includes:• Detailed information about the SDS Enterprise Database API• Supporting information such as sample code, javadoc reference, andconnectivity requirements for the database.1-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_Overview.fm

Getting Started 22In This Chapter Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2Powering On the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4Using the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7Basic Software Skills Tutorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10Using SDS Plate Documents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-29Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-1DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedGetting StartedBefore You BeginProcedureQuick ReferenceIf this is your first time using the Applied Biosystems 7900HT Fast Real-Time PCRSystem, consider completing the “Basic Software Skills Tutorial” on page 2-10before continuing. The tutorial will provide you with the fundamental knowledgerequired to operate the SDS software and will teach you timesaving techniques toallow you to use it quickly and efficiently.The following table contains a list of major procedures described inside this manual.Setting Up and Running SDS ExperimentsCreating an SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-9Running an Individual SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23Running Batches of SDS Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-31Stopping a Run from the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-28Stopping a Run from the Automation Controller Software . . . . . . . . . . . . . . . . .4-41Ejecting a Plate from the SDS Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-29Ejecting a Plate from the Automation Controller Software . . . . . . . . . . . . . . . . .4-41Analyzing Run DataAnalyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Analyzing an Absolute Quantification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5Analyzing an Relative Quantification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15Analyzing a Dissociation Curve (Melting Curve) Run . . . . . . . . . . . . . . . . . . . . .6-37Maintaining the 7900HT InstrumentChanging the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-12Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-6Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-14Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-16Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-20Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-27Verifying Instrument Performance Using a TaqMan ® RNase P InstrumentVerification Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-30Maintaining the Automation AccessoryAdjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . .7-37Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-41Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . .7-49Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-52Note: The Automation Accessory includes the Zymark ® Twister Microplate Handlerand the fixed-position bar code reader. See “Instrument Components” on page 1-3for more information.2-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Getting StartedUsing the SDSSoftwareOnline HelpThe Sequence Detection Systems Software Online Help can guide you through theprocedures for setting up, performing, and analyzing runs. To get help at any time,click (Help button) located inside the dialog box or window in which you areworking.The SDS software provides two ways to access the online help:To…access general helpget help for using a specific dialog box,plot, or featureThen…select Help > SDS Online Help.click (help button) located inside the dialogbox or window in which you are working.For MoreInformationFor information about the Applied Biosystems 7900HT Fast Real-Time PCR Systemor the SDS software, Applied Biosystems recommends the following references:• Applied Biosystems 7900HT Fast Real-Time PCR System Site PreparationGuide (PN 4317595)• Applied Biosystems 7900HT Fast Real-Time PCR System Quick Starts for:– Allelic Discrimination (PN 4351673)– Absolute Quantification (PN 4351672)– Relative Quantification (PN 4351674)• Sequence Detection Systems Software Online Help• Microsoft Windows Operating System Online Help• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-TimePCR System Administrators Guide (PN 4351669)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-3DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

7900HT FAST Real-Time PCR SystemChapter 2Getting StartedPowering On the 7900HT InstrumentPowering On theInstrumentThe activation of the Applied Biosystems 7900HT Fast Real-Time PCR System issequential. You must activate each component in a specific order for the system toinitialize properly. If performed out of sequence, the components may not be able toestablish the necessary communication connections required for operation.Status lightsGR20097900HTFront view with RobotComputerpower button7900HT instrumentpower buttonGR2009Zymark Twister MicroplateHandler (power button in rear)Monitorpower buttonFigure 2-1 Power Buttons of the Applied Biosystems 7900HT Fast Real-TimePCR SystemIMPORTANT! Power on the power to the instrument and the Plate Handler at least10 min before use. When activated, the instrument heats the sample block cover to105 °C. If you start a run before the heated cover reaches 105 °C, the instrument willpause until it reaches the optimal temperature before commencing the run.1. Power on the monitor and computer.2. Power on the Zymark Twister Microplate Handler by pressing the power switchlocated on the back panel of the Plate Handler (see below).PHYSICAL HAZARD. Keep clear of the arm whenactivating the Plate Handler. Once activated, the arm automatically moves to itshome position.HI-POTA B C DPower switchRear Panel of the TwisterGR1728If operating normally, the Plate Handler moves the arm to the home position(over the output stack).3. Power on the 7900HT instrument by pressing the power button located belowthe status lights on the front of the instrument (see Figure 2-1).If operating normally, the 7900HT instrument will do the following on startup:• Emit a high-pitched tone signaling that the instrument initialized successfully.2-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

GR23967900HTPowering On the 7900HT Instrument• Cycle the status lights (Red, Orange, Green) indicating that the 7900HTinstrument is active (see “Reading the Instrument Status Lights” on page 2-5for more information).IMPORTANT! Do not power on the 7900HT instrument if you have removed thelower-side panel of the instrument. Doing so prevents the instrument from uploadingthe firmware from the computer and causes the software to display an error.Reading theInstrumentStatus LightsThe 7900HT instrument contains three lights located on the lower-left side of thefront panel to indicate the status of the instrument (see Figure 2-2).Status lightsPower buttonFigure 2-2Status Lights of the 7900HT InstrumentTable 2-1Instrument Status LightsLight/Appearance Status Action(Green)SolidThe 7900HT instrument is onand in idle state (ready to run)Flashing • Interlocks are open and/orthe scan head has notreached the safe position.• The instrument door is open.NoneThis state indicates normalinstrument function.(Orange)FlashingSolidThe 7900HT instrument istransmitting/receiving datato/from the computer(usually during a run).If the light remains on duringstartup for more than 2 min:• The instrument did not bootproperly, or• 7900HT instrument hasexperienced a system failureNoneThis state indicates normalinstrument function.1. Check that the computer ispowered on and connectedto the instrument. (Seepage 1-10 for a diagram ofinstrument connections.)2. If so, power off theinstrument, wait for 30 sec,and then restart as explainedon page 2-4.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-5DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedTable 2-1Instrument Status LightsLight/Appearance Status Action(Red)SolidThe 7900HT instrument hasdetected a fatal problem.Power off the instrument, waitfor 30 sec, and then restart.Note: If the problem persists,contact Applied BiosystemsTechnical Support.2-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Using the SDS SoftwareUsing the SDS SoftwareStarting theSoftwareAbout theSoftwareInterface1. Do one of the following:• Select > Programs > Applied Biosystems > SDS 2.2.1 >SDS 2.2.1.• Double-click (SDS 2.2.1) on the computer desktop.If you are using an SDS Enterprise Database on your local area network, thesoftware will automatically prompt you to log in.2. (Database Only) In the Login dialog box, enter your user name and password,and click .Note: When working with a database, the SDS software requires that all usershave a user account and password (see the SDS Enterprise Database for theApplied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide(PN 4351669) for more information on assigning and modifying user accounts.)The computer starts the SDS software and attempts to establish communication withthe 7900HT instrument. If the connection is successful, the software displays theConnected icon () in the status bar when a plate document is open.See “About the Message Bar” on page 2-9 for more information.All software operations and displayed information occur inside the workspace of theSDS software. The workspace provides quick access to all elements of the softwarethrough the menubar and a pair of toolbars. Figure 2-3 summarizes the features of theuser interface of the SDS software.MenubarContains a directory of menusgoverning the operation of thesoftwareWindow buttonsMinimize ( ), maximize ( ),or close ( ) the windowGeneral ToolbarContains icons for controllingthe basic functions of thesoftware (see page 2-8)Display ToolbarContains icons for controllingthe display of information inthe SDS software workspace(see page 2-8)Message BarDisplays messages that indicatethe status of the instrument andthe SDS software (see page 2-9)Figure 2-3Elements of the SDS Software User InterfaceInstrument Connection IconIndicates the status of theconnection to the 7900HTinstrument (see page 2-9)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-7DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedAbout theGeneral ToolbarAbout theDisplay ToolbarThe general toolbar contains tools used to control the basic functions of the softwarefor data management and analysis. The following list summarizes the functions ofthe tools and provides a reference in parenthesis ( ) to a procedure involving thefunction.– Creates a new plate document (see page 2-13)– Opens a plate document from the hard drive (see page 2-19)– Saves the plate document to the hard drive (see page 2-17)– Opens a plate document from the database (see page 2-19)– Saves the plate document to the database (see page 2-17)IMPORTANT! The general toolbar will display the and options only ifthe SDS software is connected to an SDS Enterprise Database.– Imports data from a text file (see page 2-16)– Exports data to a text file (see page 2-14)– Opens the Print Report dialog box (see page 2-13)– Opens the Find tool (see page 2-13)– Removes the selected object and places it into memory (see page 2-13)– Copies the selected object into memory (see page 2-13)– Inserts the object in memory into the current selection (see page 2-13)– Analyzes the plate document (see page 2-13)– Opens the Analysis Settings dialog box (see page 2-13)The display toolbar contains tools used to control the display of information insidethe SDS software workspace. The following list summarizes the functions of thetools and provides a reference in parenthesis ( ) to a procedure involving the function.– Hides/shows the Well Inspector Panel (see page 2-12)– Hides/shows the Plate List Panel (see page 2-12)– Hides/shows the Plate Grid (see page 2-12)– Hides/shows the Table Pane (see page 2-12)– Hides/shows the System Raw Data Pane (see page 2-12)– Hides/shows the Multicomponent Plot (see page 2-12)– Hides/shows the Amplification Plot (see page 2-12)– Hides/shows the Standard Curve Plot (see page 2-12)– Hides/shows the Gene Expression Plot (see page 2-12)– Hides/shows the Dissociation Plot (see page 2-12)– Zooms the plate grid (see page 2-12)– Opens the Display Settings dialog box (see page 2-12)2-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Using the SDS SoftwareAbout theMessage BarThe message bar displays a variety of messages to indicate the status of theinstrument and software. The following table summarizes all of the messagesdisplayed in the Message bar.Table 2-2Status MessagesMessageReadyCollecting DataReanalyze dataAnalyzing data... + Progress barSaving data... + Progress barImporting data...Exporting data... + Progress barThen the SDS software is…ready and awaiting instructions.currently running a plate document.requesting analysis of plate document data.The analysis settings for the plate document havebeen changed and the document requires reanalysisfor them to take effect.completing the calculations for the current analysis.The metered bar to the right of the message displaysthe progress of the analysis.saving the plate document or plate documenttemplate to a storage device.The metered bar to the right of the message displaysthe progress of the action.importing a file.The metered bar to the right of the message displaysthe progress of the action.exporting the data inside the current plate documentto a file.The metered bar to the right of the message displaysthe progress of the action.About theInstrumentStatus IconThe Instrument Status Icon indicates the status of the connection to the 7900HTinstrument.– The computer has successfully connected to the instrumentand is awaiting a command– The computer is not able to connect to the instrument (theinstrument is not connected or is powered off)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-9DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedBasic Software Skills TutorialUser AccessRequirementAbout ThisTutorialAccessing theOnline TutorialIf using a computer linked to an SDS Enterprise Database, you must have Scientist orAdministrator access privileges to complete this tutorial.This tutorial will:• Teach you to create, save, print, export, and import SDS plate documents• Familiarize you with the basic components of the SDS software interface• Explain how to customize and arrange the user interface to suit your needs• Teach you to use the hand-held bar code reader• Provide you with timesaving techniques that will increase your effectivenesswith the SDS softwareThe Sequence Detection Systems Software Online Help provides a version of thistutorial. If you prefer to follow the online tutorial, open the online help:1. If not already active, start the SDS software as explained on page 2-7.2. Select Help > SDS Online Help.3. In the SDS Online Help, click Basic Skills Tutorial from the list of options.4. Follow the directions displayed on your screen.2-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when performingthe lessons in this tutorial. This section describes the types of actions you may needto perform depending on how your administrator has configured the database.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.If the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 2-4Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.If the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.Description of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 2-5Electronic Signature Verification Dialog Box OptionsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-11DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedLesson 1: Using Plate DocumentsAbout SDS PlateDocumentsEvery plate run on the Applied Biosystems 7900HT Fast Real-Time PCR Systemrequires the creation of a plate document using the SDS software. A plate documentis a virtual representation of a consumable used to contain samples and reagentsduring a sequence detection run. The software uses the plate document to coordinatethe operation of the instrument (thermal cycling and data collection), to organize andstore the data gathered during the PCR, and to analyze the run data.Table 2-3Plate Document Types of the SDS SoftwareFile Ext. Icon DescriptionSDS 7900HTDocumentSDS 7900HTTemplateDocumentSDS 7900HTMultiple PlateDocument*.sds*.sdt*.sdmPlate Documents are the primary file you will use.They are generated for every kind of experimentand are generally used to run plates.Although optional, plate document templates areuseful as timesaving devices for experiments wheresamples are run on plates with identical assayconfigurations.Multiple plate documents are used to analyze datafrom relative quantification experiments. The use ofmultiple plate documents is discussed in detail inChapter 6, “Analyzing Real-Time Data.”The exercises in this lesson will familiarize you with the use of plate documents.2-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialExercise 1:Creating a PlateDocumentYou will need to create a plate document for every plate you run on the 7900HTinstrument.IMPORTANT! The SDS software can handle multiple documents simultaneously,however the processing speed of your computer will decrease with each opendocument. For that reason, Applied Biosystems recommends limiting the number ofopen documents to no more than 10.1. If not already active, start the SDS software:a. Select > Programs > Applied Biosystems > SDS 2.2.1 >SDS 2.2.1).b. (Database Only) In the Login dialog box, enter your user name andpassword, and click .2. Click (or select File > New).3. Configure the New Document dialog box with the following settings:• Assay drop-down list – Select Absolute Quantification.• Container drop-down list – Select 96 Wells Clear Plate.• Template drop-down list – Select Blank Template.• Barcode field – Enter Practice.Note: Normally, you would enter a file name or bar code consisting of up to128-characters in the Barcode field, However, for the purpose of demonstratingthe software function, this tutorial instructs you to enter “Practice.”4. Click .The software displays a new plate document with appropriate attributes.5. Open the plate document template for the TaqMan ® RNase P InstrumentVerification Plate (PN 4310982, for the Standard 96-Well Block) and export itsplate setup as explained on page 2-14.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-13DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedExercise 2:Exporting Datafrom a PlateDocumentAs an alternative to setting up plate documents manually (as described in Chapter 4,Operating the Instrument), the SDS software can import plate document setupinformation directly from tab-delimited Assay Plate Setup Files. You can exportAssay Plate Setup Files from the SDS software or create them using a third-partyapplication such as spreadsheet or LIMS software (see Appendix A, “SoftwareReference,” for more information). In the following exercise, you will export theAssay Plate Setup File for a plate document template so that you will import in“Exercise 3: Importing Setup Table Data into a Plate Document” on page 2-16.Note: In addition to Assay Plate Setup Files, the SDS software can also export datafrom most of the analysis plots, graphs, and tables. See Appendix A, “SoftwareReference,” for more information.Opening the Plate Document TemplateIn the following procedure, you will open the plate document template for aTaqMan ® RNase P Instrument Verification Plate. The SDS software automaticallyinstalls several default plate document templates to:\AppliedBiosystems\SDS2.2.1\Templates. In addition to the RNase P Plate documenttemplate, Applied Biosystems provides in the same directory several plate documenttemplates for plates run regularly on the 7900HT instrument.1. Click (or select File > Open).2. In the Look In field of the Open dialog box, navigate to:\AppliedBiosystems\SDS2.2.1\Templates.3. Select File of type > SDS 7900HT Template Document (*.sdt).4. In the Look In field, select the 96 Well RNaseP Install Plate.sdt file.Select5. Click .The software opens the plate document template.6. Export the plate setup information from the plate document template asexplained on page 2-15.2-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialExporting the Plate Setup Information1. Click (or select File > Export).2. In the Look In field of the Export dialog box, navigate to:Applied Biosystems/SDS Documents.3. In the Export dialog box, select Export > Setup Table.4. Select the All Wells radio button.5. Select the SDS 2.2.1 radio button.6. In the File name field, enter Practice.7. Click .The software saves the plate document setup table information to atab-delimited text file entitled ‘Practice.txt’.Note: If desired, you can open the ‘Practice.txt’ Assay Plate Setup File usingthe Microsoft Notepad, Wordpad, or Word software to view its contents.8. Select File > Close to close the plate document template.IMPORTANT! Do not close the plate document created in “Exercise 1: Creatinga Plate Document” on page 2-13.9. Import the exported plate setup information into the plate document asexplained on page 2-16.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-15DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedExercise 3:Importing SetupTable Data into aPlate DocumentAs a timesaving device, the SDS software allows you to import plate document setupinformation from exported or fabricated Assay Plate Setup Files. To illustrate thisfeature, import the plate grid setup information contained in the Practice.txt file(from “Exercise 2: Exporting Data from a Plate Document” on page 2-14) into theempty plate document created in “Exercise 1: Creating a Plate Document” onpage 2-13.1. If the plate document from Exercise 1 is still open, go to step 2. Otherwise,create a new plate document to receive the setup table data:a. Click (or select File > New).b. Configure the New Document dialog box with the same settings as theplate document template:Assay drop-down list – Select Absolute Quantification.Container drop-down list – Select 96 Wells Clear Plate.Template drop-down list – Select Blank Template.Barcode field – Leave blank.c. Click .The software displays a new plate document with appropriate attributes.2. Click (or select File > Import).3. In the Look In field of the Import dialog box, navigate to:Applied Biosystems/SDS Documents.4. Select the Practice.txt file created in Exercise 5.5. Click .The software imports the setup information of the Practice.txt file into the plategrid and table of the empty plate document.Note: For more information on importing and exporting setup table data usingthe SDS software, see Appendix A, “Software Reference.”6. Save the plate document as explained on page 2-17.2-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialExercise 4:Saving a PlateDocumentThe Save command stores any changes to the plate document setup information anddisplay settings. If using the version of the SDS software that includes the clientsoftware for the SDS Enterprise Database, you also have the option of saving theplate document file to the database.Choose from the procedures below:• If using a database, save the plate document to the database as described onpage 2-18.• If you are not using a database, save the plate document to your computer harddrive as described below.Saving the Plate Document as an SDS 7900HT Document1. Click (or select File > Save As).2. Select File of type > SDS 7900HT Document (*.sds).3. In the File name field, enter Practice.4. Click .5. In the Document not properly set up warning, click .The software saves the plate document to a file entitled Practice.sds.6. Close the saved plate document file as explained on page 2-18.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-17DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedSaving the Plate Document to the SDS Enterprise Database1. Click (or select File > Save Document to Database).2. In the Document not properly set up warning, click .3. In the Save Document to database dialog box, leave the Comment field blank orenter a brief comment describing the file.Note: The Comment field can contain up to a 256-character description of thefile.4. Click to save the file.5. In the Document not properly set up warning, click .6. In the Saved Document dialog box, click .7. Select File > Close.8. If prompted to save the plate document, click .The SDS software closes the file without saving it.9. Open a plate document file as explained on page 2-19.2-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialExercise 5:Opening a PlateDocumentIn this exercise, you will be opening two documents that you will use in the followingexercises: the plate document (from Exercise 3), and a plate document template. Thepurpose of this lesson is to familiarize you with the tasks of retrieving plate documentthat have been saved to the computer hard drive or the SDS Enterprise Database.Choose from the procedures below:• If using a database, save the plate document to the database as described onpage 2-18.• If you are not using a database, save the plate document to your computer harddrive as described below.Opening the SDS Plate Document File1. Click (or select File > Open).2. In the Look In field of the Open dialog box, navigate to:Applied Biosystems/SDS Documents3. In the Look In field, select the Practice.sds file.Select4. Click .The software opens the Practice.sds file.5. Select File > Close.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-19DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedOpening the Plate Document from the SDS Enterprise Database1. Click (or select File > Open Document from Database).2. Configure the Find Plates Matching These Criteria table of the Plate Querydialog box:a. Click the first cell in the Variable column, and select Barcode.b. Click the first cell in the Conditions column, and select Contains.c. Click the first cell in the Value1 column, and enter Practice.3. Click .SelectBarcodeSelectContainsEnterPracticeClick to begin the search4. In the Search Results list, select the Practice plate document, and clickClick to selectthe documentClick to open the document5. Click .6. Go on to “Lesson 2: Viewing and Resizing Panes” on page 2-21.2-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialLesson 2: Viewing and Resizing PanesOverviewExercise 1:Resizing Panes,Views, and PlotsBecause plate documents can display setup and analysis data in multiple viewssimultaneously, the SDS software provides several navigational devices to helpmanage the information. This lesson will teach you to use the different aids to reducescreen clutter and ensure efficient use of the software.You can resize the panes, views, and plots of plate documents by moving the greylines dividing them horizontally and vertically.Dividing line(click and drag)Dividing line (click and drag)1. Click and drag the grey line dividing the plate grid and well inspector to the right.The software expands the plate grid and table pane to the new width.2. Using the grey dividing lines, resize other elements of the plate document untilyou are comfortable using the feature.Exercise 2: Hidingand ShowingPanes, Views,and PlotsYou can also toggle the presence of the plate document panes, views, and plots usingthe icons in the Display toolbar.1. In the Display toolbar, click (the Hide/Show Table Pane button).The software removes the table pane from the plate document.2. Click again to show the table pane.The software restores the table pane to the plate document.The display toolbar can be particularly useful for manipulating informationshown in the plate document. See “About the Display Toolbar” on page 2-8 for alist of the other icons of the display toolbar and the panes they control.3. Practice hiding and showing the other plate document elements by clickingother buttons in the Display toolbar until you are comfortable using the feature.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-21DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedExercise 3:Maximizing/MinimizingPanes, Views,and PlotsYou can maximize the panes, views, and plots of plate documents by clicking thesizing buttons embedded inside the grey dividing lines.Note: Sizing buttons are the arrowhead marks ( ) that appear between adjacentelements of the plate document. When clicked, a sizing button hides the element towhich it points.1. Click (down-arrow sizing button) in the divider between the plate grid and thetable pane to maximize the plate grid vertically.The software maximizes the plate grid by minimizing the table pane.Sizing button2. Click and drag the grey divider at the bottom of the plate document to restore thetable pane to its original size (using the action described in Exercise 1).3. Using the sizing buttons, maximize/minimize other elements of the platedocument until you are comfortable using the feature.2-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialLesson 3: Using the Plate GridOverviewThe plate grid (see Figure 2-6) is an important interface tool for the SDS software.The software uses the grid to convey information about the plate and allows you toselect specific wells for viewing and analysis. The following exercises will teach youhow to use and modify the elements of the plate grid.Plate gridFigure 2-6Plate Grid of SDS Plate DocumentsExercise 1:Viewing WellInformationThe SDS software provides two methods for viewing the information associated witha well or wells of the plate document. To view the information for a well of the platedocument, do one of the following:• Click any well in the plate grid to select it.When selected, the software outlines the well in black and displays theassociated information in the well inspector of the Setup tab.Setup tab displays theinformation of the selected well• Move the pointer over any well of the plate grid.The software displays the information for the well in a yellow pop-up window.Pop-up displays the informationof the well to which the mousecursor pointsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-23DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedExercise 2:Selecting MultipleWellsThe SDS software provides several methods for selecting wells from the plate grid.The following exercise will familiarize you with most of them.1. Select a block of wells from the plate grid by doing one of the following:• Click and drag the mouse cursor across the block of wells, or• Click the well at the top-left corner of the block. Then while holding-downthe Shift key, click the well at the bottom-right corner of the block.The software outlines the selected wells with a black border.2. Select several isolated wells of the plate grid by doing of the following:a. Hold-down the Ctrl key, and click individual wells to select them.The software outlines the selected wells with a black border.b. While holding down the Ctrl key, click the wells to de-select them.2-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills Tutorial3. Select an entire column or row of wells using the headers:a. Click the header for row B to select all wells in the row.The software outlines the wells of row B with a black border.b. Press and hold either the Shift or Ctrl key, then click other columns or rowheaders to select multiple columns.Row B headeror4. Select all wells of the plate grid by clicking the top-left corner of the plate grid.The software outlines all of the wells in the plate document with a black border.Button5. Using the techniques illustrated in steps 1 to 4, practice selecting portions of theplate grid until you are comfortable using the feature.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-25DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedExercise 3:Zooming thePlate GridYou can zoom the plate grid to display the well information by clicking the ZoomGrid button ( ).1. Click and observe how the grid expands to display the well information.2. Click again to restore the plate grid to the original size.3. Practice zooming portions of the plate grid until you are comfortable using thefeature.Exercise 4:Resizing WellsUsing the BorderLinesYou can also adjust the size of the plate grid wells by moving the lines in the row orcolumn headers.1. Move the mouse cursor over a border line in the row or column header.The mouse cursor becomes a double arrow ( ).2. Click and drag the mouse cursor to adjust the well to a new width.The software resizes all wells in the plate grid to match the new width.Mouse cursor3. Practice resizing the wells of the plate grid until you are comfortable using thefeature.2-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Basic Software Skills TutorialLesson 4: Using the Hand-Held Bar Code ReaderOverviewExercise:Entering BarCode InformationUsing theHand-Held BarCode ReaderThe hand-held bar code reader functions as an extension of the keyboard that you canuse to automatically enter bar codes into the SDS software. When the reader is usedsuccessfully to scan a bar code, it automatically:• Transmits the alphanumeric equivalent of the bar code to the software. Thesoftware enters the bar code text wherever the cursor is active.• Transmits a carriage-return (the equivalent of pressing the Enter key).The following procedure explains how to enter a bar code number using the had-heldbar code scanner. Normally, you would scan the bar code into the New Documentdialog box during plate document creation.1. Select Tools > Document Information.2. In the Document Information dialog box, click the Comments field.Click here3. While holding the hand-held bar code reader 20 to 30 cm away from a plate, aimat the center of the bar code and press the trigger. The scanner emits a sweepinglaser beam that appears as a red line on the plate. Slowly move the scanningbeam slowly across the bar code until the scan gun emits a high-pitched toneacknowledging that it has read the code.LASER HAZARD. Exposure to direct or reflected laserlight can burn the retina and leave permanent blind spots. Never look into thelaser beam. Remove jewelry and anything else that can reflect the beam intoyour eyes. Protect others from exposure to the beam.PECY001DL320-30 cmGR2110After the gun has read the bar code, the software automatically populates theselected field with the alphanumeric equivalent of the bar code.4. Click to close the Document Information dialog box.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-27DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedLesson 5: Using Contextual MenusOverviewThe SDS software features contextual menus as a timesaving device to provideaccess to the commands for an associated view or pane.To access a contextual menu, move the pointer over a pane or view of interest, thenright-click. The menu appears at the location of the pointer.Contextual menuFigure 2-7Contextual Menu of the SDS SoftwareAll contextual menus provide the following common commands:Table 2-4Standard Commands of the Contextual MenuCommand Result See PageHide Hides the pane or view. 2-21Save to Image FileDisplay SettingsOpens the Export Graphic dialog box for exporting theselected view or pane as a JPEG graphic file.Opens the Display Settings dialog box, which allowsyou to modify the appearance of the view, pane, or plot.A-163-24Lesson 6: Using Keyboard ShortcutsExercise: Closingthe PlateDocumentThe SDS software provides keyboard shortcuts for invoking the major functions of thesoftware. A keyboard shortcut is a combination of two keys (Ctrl and another key)that, when pressed in unison, instruct the software to perform an action. The softwarelists the keyboard shortcuts next to the options in each menu on the menu bar.To demonstrate the use of keyboard shortcuts, close the open plate document as follows:1. Simultaneously press the Ctrl and W keys (Ctrl+W).2. When prompted to save the plate document, click .Note: The Sequence Detection Systems Software Online Help contains a completelist of the keyboard shortcuts for the SDS software. To view the list, open the onlinehelp as explained in “Using the SDS Software Online Help” on page 2-3.2-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Using SDS Plate DocumentsUsing SDS Plate DocumentsUsing MultiplePlate DocumentsElements of aPlate DocumentAlthough the SDS software can handle multiple documents simultaneously, theprocessing speed of the computer decreases with each open document. Consequently,Applied Biosystems recommends limiting the number of open documents to 10.Figure 2-8 shows a typical plate document and identifies its components which aredescribed on the following pages.Plate grid(see below)Tabs(page 2-30)Well inspector(page 2-31)Table pane(page 2-29)Figure 2-8Common Elements of SDS Plate DocumentsPlate GridTable PaneThe cells of the plate grid correspond to the wells of the reaction device that the platedocument models. The grid displays well information according to the type of platedocument. The Plate Grid properties settings in the Display Settings dialog boxdetermine content and appearance of the information displayed inside the cells of thegrid.Note: For more information on configuring the display settings for the plate grid,click (help button).The table pane displays the setup and analysis properties for the plate document. Youcan export the table pane as a tab-delimited text file for use by a spreadsheetapplication (see Appendix A, “Software Reference,” for more information).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-29DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting StartedTabsPlate documents can have up to six tabs (depending on their function) as describedbelow:Table 2-5Tabs of the SDS SoftwareTab Function Views/PlotsSetupInstrumentRaw DataPlotCalibrationDataResultsDissociationCurveDisplays well information, and allows youto configure the plate grid with setupinformation.Used to program the plate documentmethod, run the plate document, or send itto the Plate Queue.Displays the raw fluorescence collectedfrom the sequence detection run.The Raw Data tab is visible only in platedocuments containing run data.To view the tab, open a plate documentcontaining run data, click , then selectthe Raw Data Plot tab.Displays the Background and Pure Spectracalibration data used for the signalnormalization and multicomponentinganalysis of the run data.The Calibration Data tab is visible only inplate documents containing run data.To view the tab, open a plate documentcontaining run data, click , then selectthe Calibration Data tab.Displays analyzed run data.The Analysis tab is visible only in platedocuments containing analyzed run data.Displays analyzed dissociation curve datafrom a programmed ramp.The tab is visible only in plate documentscontaining analyzed data from a real-timerun with a programmed ramp.Well Inspector• Method Editor– Thermal Profile tab– Auto Increment tab– Ramp Rate tab– Data Collection tab• Real-Time tab• Plate-Read tabRaw Data PlotCalibration Data• Background Plot• Pure Dyes PlotAllelic Discrimination Data• Allelic Discrimination PlotAbsolute Quantification• Amplification Plot• Standard (Curve) PlotRelative Quantification• Amplification PlotDissociation Plot2-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Using SDS Plate DocumentsWell InspectorThe Well Inspector (Figure 2-9) applies detector and sample information to the wellsinside the grid pane and displays information from the selected cells in the plate grid.Sample name fieldDetector listPassive Reference drop-down listOmit Well check boxFigure 2-9Components of the Well Inspector• Sample Name text field – An editable field that displays the sample nameapplied to the selected well(s)Note: The Sample Name field will display *Mixed* if multiple wells withdifferent sample names are selected.• Detector list – Lists all available detectors copied to the plate document• Omit Well check box – Toggles the activity of the well. If selected, the softwareeliminates the data from the selected well from all analysis procedures.• Passive Reference drop-down list – Displays the fluorescent dye used as apassive referenceApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-31DRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Chapter 2Getting Started2-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, CH_GetStart.fm

Preparing a Run 33In This Chapter Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5Quick Review: Powering On the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . . . 3-8Step 1 – Creating a Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9Step 2 – Applying Detectors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11Step 3 – Configuring the Plate Document with Tasks . . . . . . . . . . . . . . . . . . . . . 3-16Step 4 – Setting the Passive Reference and Omitting Wells . . . . . . . . . . . . . . . . . 3-18Step 5 – Programming the Plate Document Method. . . . . . . . . . . . . . . . . . . . . . . 3-19Step 6 – Saving the Plate Document as a Template. . . . . . . . . . . . . . . . . . . . . . . . 3-24Step 7 – Creating a Plate Document from the Template . . . . . . . . . . . . . . . . . . . . 3-26Step 8 – Applying Sample and Plate Information. . . . . . . . . . . . . . . . . . . . . . . . . 3-28Step 9 – Running the Plate on the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . 3-29Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-1DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when creating andmodifying plate documents as described in this chapter. This section describes thetypes of actions you may need to perform depending on how your administrator hasconfigured the database. For more information on any of the features describedbelow, see the SDS Enterprise Database for the Applied Biosystems 7900HT FastReal-Time PCR System Administrators Guide.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.When the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 3-1Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.When the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.3-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Notes for Database UsersDescription of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 3-2Electronic Signature Verification Dialog Box OptionsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-3DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunBefore You BeginBackgroundInformationGetting MoreInformation fromthe SDSOnline HelpMaximizingThroughput forEnd-Point RunsChapter 5, “Analyzing End-Point Data,” and Chapter 6, “Analyzing Real-Time Data,”include brief discussions of the experiments that you can perform using the 7900HTinstrument. Before beginning, you may want to review the appropriate chapter foryour experiment:Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Absolute Quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-37The Sequence Detection Systems Software Online Help can guide you through theprocedures for setting up, performing, and analyzing runs. To get help at any time,click the button located inside the dialog box or window in which you areworking.For end-point applications such as allelic discrimination, the throughput of theApplied Biosystems 7900HT Fast Real-Time PCR System can be increased bydividing the workload between the 7900HT instrument and several thermal cyclers.Unlike real-time runs, the 7900HT instrument collects data for end-point runs afterthe completion of the PCR. Consequently, you can perform the thermal cycling ofend-point plates elsewhere and then transfer them to the 7900HT instrumentafterwards for data collection and analysis.IMPORTANT! To perform the thermal cycling and the plate read using the 7900HTinstrument, run the plate first as a real-time plate document and then again as anallelic discrimination plate document (see “Step 5 – Programming the PlateDocument Method” on page 3-19 for the procedure).3-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Workflow OverviewWorkflow OverviewExperiments/Runs Performedon the 7900HTInstrumentAbsoluteQuantificationWorkflowAbsolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Dissociation Curve (Melting Curve) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6Pure Dye (Spectral Calibration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6RNase P Instrument Performance Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-301. Create an absolute quantification plate document (see page 3-9).2. * Apply detectors to the plate document:a. Create detectors for the absolute quantification probes (see page 3-11).b. Copy the detectors to the plate document (see page 3-13).3. Configure the plate document with tasks and quantities:a. Configure the plate document with detector tasks (NTC, Standard, andUnknown) (see page 3-16).b. Assign quantities to the wells of the plate document that contain standards(see page 3-17).4. Program the method for the absolute quantification run (see page 3-19).5. If performing an assay in which you would like to collect dissociation data, add atemperature ramp to the thermal profile to perform a dissociation curve analysis(see page 3-23).6. Choose from the following:– If running a single plate, then continue to step 2.– If running the first plate in a series of plates with identical assayconfigurations, then save the plate document as a template (see page 3-24).7. Create a plate document from the template created in step 2 (see page 3-26).8. Configure the document with sample names and plate information (seepage 3-28).9. Prepare and run the absolute quantification plate or plates (see page 4-1).10.Analyze the run data (see page 6-5).*Steps 2 and 2 can be eliminated by importing the plate document setup informationfrom a tab-delimited text file. See “Importing Plate Document Setup Table Files”on page A-2 for more information.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-5DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunRelativeQuantificationWorkflow1. Create a relative quantification plate document (see page 3-9).2. * Apply detectors to the plate document:a. Create detectors for the relative quantification probes (see page 3-11).b. Copy the detectors to the plate document (see page 3-13).3. * Configure the plate document with detector tasks (NTC and Unknown)(see page 3-16).4. * Program the method for the relative quantification run (see page 3-19).5. Choose from the following:– If running a single plate, then continue to step 2.– If running the first plate in a series of plates with identical assayconfigurations, then save the plate document as a template (see page 3-24).6. Create a plate document from the template created in step 2 (see page 3-26).7. Configure the document with sample names and plate information (seepage 3-28).8. Prepare and run the relative quantification plate or plates (see page 4-1).9. Analyze the run data (see page 6-15).*Steps 2 and 2 can be eliminated by importing the plate document setup informationfrom a tab-delimited text file. See “Importing Plate Document Setup Table Files”on page A-2 for more information.Dissociation(Melting) CurveWorkflowThe SDS software can perform a dissociation curve analysis as part of an absolutequantification run. Therefore, to perform a dissociation curve, construct a platedocument for absolute quantification as explained on page 3-5 and configure themethod with a temperature ramp as explained on page 3-23.3-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Workflow OverviewAllelicDiscriminationWorkflow1. Create an allelic discrimination plate document (see page 3-9).2. * Apply markers to the plate document:a. Create detectors for the allelic discrimination probes (see page 3-11).b. Create a marker for each allelic discrimination probe pairing (see page 3-14).c. Copy the marker(s) to the plate document (see page 3-15).3. * Assign detector tasks to the wells of the plate document (NTC and Unknown)(see page 3-16).4. If you would like to perform thermal cycling of the allelic discrimination plate onthe 7900HT instrument, create a real-time plate document for the plate andprogram it with the method for the allelic discrimination run. Otherwise, continueto step 2 (see page 3-19).5. Choose from the following:– If running a single plate, then continue to step 2.– If running the first plate in a series of plates with identical assayconfigurations, then save the plate document as a template (see page 3-24).6. Create a plate document from the template created in step 2 (see page 3-26).7. Configure the document with sample names and plate information(see page 3-28).a. Prepare the allelic discrimination plate or plates and perform thermal cyclingon a designated thermal cycler (see page 4-1).b. Run the allelic discrimination plate or plates on the 7900HT instrument.8. Analyze the run data (see page 5-5).*Steps 2 and 2 can be eliminated by importing the plate document setup informationfrom a tab-delimited text file. See “Importing Plate Document Setup Table Files”on page A-2 for more information.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-7DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

GR23967900HTChapter 3Preparing a RunQuick Review: Powering On the 7900HT InstrumentPowering On theInstrumentNote: The following table includes a set of abridged procedures for activating thecomponents of the Applied Biosystems 7900HT Fast Real-Time PCR System. For acomplete explanation of the procedure, see “Powering On the 7900HT Instrument”on page 2-4.IMPORTANT! Do not power on the 7900HT instrument while the lower-side panel ofthe instrument is removed. Doing so prevents the instrument from uploading thefirmware from the computer and causes the software to display an error.1. Power on the monitor and computer.2. If using an automation module, power on the Zymark ® Twister MicroplateHandler.HI-POTA B C DPower switch(Rear panel of the Twister)GR17283. Power on the 7900HT instrument.Power button4. Start the SDS software (select > Programs >Applied Biosystems > SDS 2.2.1 > SDS 2.2.1)5. If using the SDS Enterprise Database, enter your user name and password in theappropriate fields of the Login dialog box, then click .The SDS software displays the program window.3-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 1 – Creating a Plate DocumentStep 1 – Creating a Plate DocumentUser AccessRequirementAbout ABI PRISMPlate DocumentsCreating a PlateDocumentIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to create plate documents.Every plate run on the 7900HT instrument requires the creation of a plate documentwithin the SDS software. Each plate document is a virtual representation of aspecific consumable (that is, a reaction plate or Low Density Array) containingsamples and reagents for use on the 7900HT instrument.Plate documents contain the following information:• Detector information and arrangement on the plate• Marker information and arrangement on the plate (allelic discrimination only)• Sample information and arrangement on the plate• Method parameters for the run (absolute and relative quantification)1. If not already open, start the SDS software as explained on page 3-8.2. Click (or select File > New).3. Configure the New Document dialog box with settings for the run:• Assay drop-down list – Select the type of assay appropriate for your plate.• Container drop-down list – Select the type of consumable you intend to run.• Template drop-down list – Select Blank Template.• Barcode field – Do one of the following:If you are creating a:– Plate document to run a single plate, scan or enter the bar code for the plate.– Plate Document Template for creating multiple plates, leave the fieldblank.IMPORTANT! The SDS Enterprise Database does not support the creation oftwo plate documents of different run types with the same bar code.Note: If performing a dissociation curve experiment, select AbsoluteQuantification from the Assay drop-down list.Assay drop-down listContainer drop-down listTemplate drop-down listBarcode field4. Click .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-9DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a Run5. Create and copy detectors (and markers) to the new plate document as describedin “Step 2 – Applying Detectors and Markers” on page 3-11.3-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 2 – Applying Detectors and MarkersStep 2 – Applying Detectors and MarkersUser AccessRequirementCreatingDetectorsIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to create and apply detectors and markers.Before you can use a plate document to run a plate, it must be configured withdetector information for the experiment (and marker information if performingallelic discrimination). A detector is a virtual representation of: a TaqMan ® probe andprimer set used for detection of a single target nucleic acid sequence, or a primer setutilizing SYBR ® Green Double-Stranded DNA Binding Dye 1 (used for the detectionof double-stranded DNA product). Before using the plate document, you must createand apply detectors for all assays present on the plate.1. Select Tools > Detector Manager.2. In the Detector Manager dialog box, click .3. Configure the Add Detector dialog box:• Name field – Enter a name for the detector.The name of the detector must be unique and should reflect the target locusof the assay (such as GAPDH or RNase P). Do not use a name for morethan one detector. The SDS software does not distinguish betweendetectors of the same name, even if they use a different dye set.• Group field – (Optional) Enter or select a detector group for the detector.• Description field – (Optional) Enter a brief description of the assay(up to 32 characters).• Reporter/Quencher drop-down lists – Select the appropriate reporter andquencher dyes for the probe.If creating a detector for an assay using the SYBR Green 1 dsDNA BindingDye, set the Quencher Dye drop down list to Non Fluorescent.If you are using a custom dye not manufactured by Applied Biosystems,you must create and run a pure dye plate for the dye before applying it to adetector (see “Adding Custom Dyes to the Pure Dye Set” on page 7-27).• Color box – (Optional) Click the box, then use the Color Picker dialog boxto select a color to represent the detector, and click .• Notes field – (Optional) Enter any additional comments for the detector(up to 200 characters).Name fieldGroup fieldDescription fieldReporter/Quencher drop-down listsColor boxNotes fieldCreation and Modificationdate stampsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-11DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunAllelic DiscriminationDetectorAbsolute or RelativeQuantification Detector4. Click .The software saves the new detector and displays it in the detector list.Note: If using the SDS Enterprise Database, you cannot create a detector thatthat uses the same combination of detector name, reporter dye, and quencherdye as an existing detector.5. Repeat steps 2 through 4 to create detectors for all remaining assays on the plate.Note: Click the button for information on the Detector Manager dialog boxor to view the procedures for editing, deleting, or searching for detectors.6. Choose from the following:If constructing aplate document for…• absolute quantification• relative quantification• dissociation curve analysisallelic discriminationThen…copy the detector(s) to the plate document asexplained in the procedure on page 3-13.create and apply markers to the platedocument as explained on page 3-14.3-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 2 – Applying Detectors and MarkersCopyingand ApplyingDetectors to thePlate DocumentIMPORTANT! Once you copy a detector to the plate document, it is no longer linkedto the corresponding entry in the Detector Manager. Consequently, if you modify adetector using the Detector Manager after you have copied it to a plate document,you must remove the detector from the plate document and copy it again to updatethe plate document with the changes.1. In the Detector Manager dialog box of the SDS software, copy the detectors tothe plate document:a. While pressing and holding the Ctrl key, select the detectors you want toapply to the plate document.The software highlights the selected detectors.b. Click .The software adds the detectors to the well inspector of the plate document.Plate documentCopied detectorDetector ManagerDetector selectedfor copying2. Click to close the Detector Manager.3. In the plate grid, select the wells containing the assay for the first detector.Note: For easier selection of plate grid wells, use the Ctrl and Shift keys toselect wells individually or in groups. See page 2-23 for more information.4. Apply detector to the selection by clicking the check box for the detector in theUse column of the well inspector.Detector added toselected wells of theplate documentUse check box5. Repeat steps 3 through 4 to apply the remaining detectors to the plate grid.6. Configure the plate document with detector tasks as explained in“Step 3 – Configuring the Plate Document with Tasks” on page 3-16.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-13DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunCreating Markersfor AllelicDiscriminationAllelic discrimination plate documents feature the use of ‘markers’ to aid inorganizing and applying detectors based on the loci they target. A marker is a pairingof two detectors representing chemical assays designed to discriminate betweendifferent alleles of a common locus. The SDS software uses marker informationduring data analysis to organize and compare the processed run data.IMPORTANT! Allelic discrimination plate documents must contain at least onemarker.1. If the Detector Manager dialog box is open, click to close it.2. Select Tools > Marker Manager.3. In the Marker Manager dialog box, click .4. In the Enter name of new Marker field of the Add Marker dialog box, enter aname for the new marker, and click .The new marker appears in the Markers field.Note: If using the SDS Enterprise Database, you cannot create a marker thatthat uses the same combination of marker name and detectors as an existingmarker.5. Apply detectors to the new marker:a. In the Markers field, click the new marker to select it.The software highlights the selected marker.b. In the Detectors list, select the Use check boxes of the detectors that youwant to assign to the marker.The software highlights the selected detector.IMPORTANT! You cannot assign more than two detectors to a marker and thedetectors cannot use the same reporter dye.Detectors assignedto ‘CYP 2C9*2’6. If evaluating multiple loci, repeat steps 3 through 5 to create additional markersas needed.IMPORTANT! You must configure a marker with two detectors before you canapply it to a plate document.Note: Click for information on the features of the Marker Manager dialogbox or to delete markers from the Markers list.7. Apply the marker(s) to the allelic discrimination plate document as explained in“Copying and Applying Markers” on page 3-15.3-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 2 – Applying Detectors and MarkersCopying andApplying MarkersIMPORTANT! Once you copy a marker to the plate document, it is no longer linked tothe corresponding entry in the Marker Manager. Consequently, if you modify a markerusing the Marker Manager after you have copied it to a plate document, you mustremove the marker and copy it again to update the plate document with the changes.1. In the Marker Manager dialog box of the SDS software, copy the allelicdiscrimination marker to the plate document:a. While pressing and holding Ctrl, select the marker(s) you want to apply tothe plate document.b. Click to copy the markers to the plate document.c. In the Copy Markers To Plate dialog box, click .Markers copied tothe plate documentd. Repeat steps a and c to copy additional markers to the allelicdiscrimination plate document as needed.2. Click to close the Marker Manager dialog box.3. Select the wells containing the assays for a marker you configured in theprevious procedure.Note: For easier selection of plate grid wells, use the Ctrl and Shift keys toselect wells individually or in groups. See page 2-23 for more information.4. In the well inspector, click the Use check box of the marker you want to add tothe selected wells.Note: The detectors associated with the marker are automatically applied to theselected wells when the marker is placed in Use.‘PDAR CYP 2C9*2’added to selectedwells of the platedocumentDetectors for the‘PDAR CYP 2C9*2’markerUse check box for‘PDAR CYP 2C9*2’(selected)5. If necessary, repeat steps 3 through 4 to assign any remaining markers to theplate document.6. Configure the plate document with detector tasks as explained in“Step 3 – Configuring the Plate Document with Tasks” on page 3-16.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-15DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunStep 3 – Configuring the Plate Document with TasksUser AccessRequirementAbout DetectorTasksApplyingDetector TasksIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to apply or modify detector task settings.You must assign a ‘task’ to the detectors or markers applied to each well of the platedocument that defines their specific purpose or function on the plate. The SDSsoftware uses the detector task assignments to determine how to treat the dataproduced by the wells when analyzing the run data. Detector tasks vary depending onthe type plate document.1. Using the Ctrl and Shift keys, select the wells of the plate grid containingsamples for a particular task described in the table below.Table 3-1Detector Tasks of the SDS SoftwareExperiment Task Apply to…AllelicDiscriminationAbsoluteQuantificationRelativeQuantificationUnknownNTCUnknownStandardNTCTargetEndogenousControlall detectors of wells that contain PCR reagentsand test samples.all detectors of negative control wells that containreagents for the PCR, but lack template.all detectors of wells containing PCR reagents andtest samples for quantification.the appropriate detectors of wells that containPCR reagents and samples of known quantities.(See page 3-17 for instructions.)all detectors of negative control wells that containPCR reagents, but lack template.all detectors of wells that contain PCR reagents forthe amplification of target sequences.all detectors of wells that contain reagents for theamplification of the endogenous control sequence.2. In the well inspector, click the field in the Task column for each detector entry,and select the appropriate task from the drop-down list. The SDS software labelsall selected wells with the task.Selected wells(task appliedto selection)Task drop-down list3-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 3 – Configuring the Plate Document with Tasks3. Repeat steps 1 through 2 to apply any remaining tasks to the plate document.4. Choose from the following options:If constructing aplate document for…absolute quantification• allelic discrimination• relative quantification• dissociation curve analysisThen…assign quantities to the standard wells of the platedocument as explained below.• If necessary, set the passive reference for theplate document as explained on page 3-18.• Otherwise, program the method for your run asexplained on page 3-19.AssigningStandards forAbsoluteQuantificationFor the SDS software to create a standard curve for quantification of unknownsamples, you must apply the Standard task and quantity values to your absolutequantification plate document.1. In the plate grid of the SDS software, select the replicate wells containing thefirst standard in the dilution series.2. In the well inspector, click the field in the Task column for the appropriatedetector entry, and select the Standard from the drop-down list.3. In the well inspector, click the field in the Quantity column for the appropriatedetector, enter a quantity or concentration for the standard in the appropriateunit of measurement (starting copy number, picograms, nanograms, etc.), andpress Enter.The software labels the selected standard wells with the specified quantity.Selected wells(quantity appliedto selection)Quantity field4. Repeat steps 1 through 3 to configure the plate with the other sets of replicatestandard wells on the plate.When finished, the plate grid should contain a complete set of replicate wellslabeled with the Standard task and assigned quantities that the software willuse to compute the standard curve for the run.5. Do one of the following:• If necessary, set the passive reference for the plate document as explained on“Step 4 – Setting the Passive Reference and Omitting Wells” on page 3-18.• Otherwise, program the method for your run as explained in“Step 5 – Programming the Plate Document Method” on page 3-19.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-17DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunStep 4 – Setting the Passive Reference and Omitting WellsUser AccessRequirementSettingthe PassiveReferenceIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to change the passive reference or omit wells from use.If using an Applied Biosystems chemistry, use the default Passive Reference setting(Applied Biosystems chemistries use the ROX passive reference dye molecule). Ifrunning a custom chemistry, select a dye to use as a passive reference for the run.Note: Applied Biosystems recommends using a passive reference to normalize thesignals from the reporter dyes.1. In the Passive Reference drop-down list, select the appropriate reference dye.Passive Reference drop-down listSelect the appropriatepassive reference2. If necessary, omit wells from use as explained below. Otherwise, program themethod for the run as explained on page 3-19.Omitting Wellsfrom UseThe SDS software allows you to omit wells from use before or after running a plate.IMPORTANT! If you remove a well removed from use before you run a platedocument, the SDS software will not collect data for the well during the run.However, if you remove a well from use after you run a plate document, the softwareexcludes the well from the analysis but does not delete the data.1. While pressing and holding the Ctrl key, select the well(s) in the grid that youwant to remove.2. In the well inspector of the Setup tab, click the Omit Well(s) check box.Selected wells(removed from use)Omit Well(s) check box(selected)3. Program the method for the run as explained on page 3-19.3-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 5 – Programming the Plate Document MethodStep 5 – Programming the Plate Document MethodUser AccessRequirementAbout SDSMethodsIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to modify the method of a plate document.During a run, the SDS software controls the instrument based on the instructionsencoded in the method of the plate document. Each new plate document(except allelic discrimination) contains a default method that you must customize foryour experiment.Methods contain the:• Thermal Cycler Conditions• Auto Increment Values• Ramp Rates• Data Collection Options• Reaction Volume SettingTo create a method for…• absolute quantification• relative quantificationThen…program the method as explained on page 3-21.IMPORTANT! If using the SDS Enterprise Database, readthe discussion of considerations below.allelic discrimination see page 3-20.dissociation curve analysisTo perform a dissociation curve analysis do the following:1. Program the method for the absolute quantificationexperiment as explained on page 3-21.2. Add a temperature ramp to the method for dissociationcurve analysis as explained on page 3-23.DatabaseConsiderationsConfigure the method of your plate document to collect data only in the necessarystages of the PCR. Because of memory constraints, the SDS Enterprise Databasecannot save real-time plate documents greater than 40 MB. The file size for areal-time run depends on the length of the run and the configuration and placementof the data collection icons. Longer runs and runs configured to collect data atmultiple stages of the method can be considerably larger than the 15–25 MB average.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-19DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunProgrammingMethods for AllelicDiscriminationTo maximize instrument throughput, the SDS software does not provide the option tothermal cycle allelic discrimination plate documents. Because allelic discriminationexperiments are end-point runs that do not require data collection during the PCR,you can perform thermal cycling on a dedicated thermal cycler and then transfer tothe 7900HT instrument for data collection and analysis.If you want to thermal cycle theallelic discrimination plates on…a designated thermal cyclerthe 7900HT instrumentThen…go on to “Step 8 – Applying Sample and PlateInformation” on page 3-28follow the procedure below.Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT InstrumentTo perform the thermal cycling and the plate read using the 7900HT instrument, runthe plate first as a real-time plate document and then again as an allelicdiscrimination plate document as explained below.IMPORTANT! Follow the procedure below only if you intend to perform the PCR onthe 7900HT instrument. Otherwise, perform the PCR on a dedicated thermal cyclerand then transfer the plate to the 7900HT instrument for data collection.1. Start the SDS software.2. Create a real-time plate document for absolute quantification as described onpage 3-9.Note: It is not necessary to configure the plate document with detectors.3. Program the plate document method with the thermal cycling times andtemperatures for your protocol as described in “Programming Methods forAbsolute or Relative Quantification” on page 3-21.4. Save the plate document as explained on page 4-24.IMPORTANT! Do not save the real-time plate document to the database. Thedatabase cannot store plate documents of different run types with the same barcode.5. Run the plate using the real-time plate document as described on page 4-25.Note: Although large, the real-time file may be helpful in diagnosing andtroubleshooting the experiment later if the data from the allelic discriminationrun produces unexpected results.6. When the run is complete, close the absolute quantification plate document.7. Go on to “Step 6 – Saving the Plate Document as a Template” on page 3-24.3-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 5 – Programming the Plate Document MethodProgrammingMethods forAbsolute orRelativeQuantificationNote: The following procedure describes how to configure only the basic features ofthe method: thermal cycler conditions, sample volume, and data collection options.To further customize the method for the plate document, click in the Instrumenttab and refer to the online help for instructions on configuring the auto increment andramp rate values.1. In the SDS software, select the Instrument tab of the plate document.2. If necessary, select or de-select the 9600 Emulation check box.Note: When the 9600 Emulation check box is checked, the SDS softwarereduces the ramp rate of the 7900HT instrument to match that of theABI PRISM ® 7700 Sequence Detection System instrument.3. Modify the default thermal profile for the method as needed:To…adjust stepparameters(time/temp)Then…select a field value, enter a new value, and click anywhereoutside of the field.Temperature field(4 to 99.9 °C)Time field(0:01 to 98:59 min)add a hold, cycleset, or step1. Click the step to the left of the location you want to place thenew stage.2. Click or .The software inserts the stage into the thermal profile.Note: To add a step to a stage, select the step to the left of thelocation you want to place the step and click .Selected stepNew stepappears hereremove a step1. Click the step you want to remove.The software highlights the selected step.2. Click to remove the step from the profile.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-21DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a Run4. Configure the data collection options for the method:a. Select the Data Collection tab.b. Click below each plateau or ramp inside the cycle stage of the thermalprofile to place a data collection icon at each step.Data collection iconsIMPORTANT! Configure the method of your plate document to collect data onlyin the necessary stages of the PCR. The file size for a real-time run depends onthe length of the run and the configuration and placement of the data collectionicons. Longer runs and runs configured to collect data at multiple stages of themethod can be considerably larger than the 15–25 MB average.5. Click the Sample Volume (µL) field and enter the volume of the reactions onthe plate.Note: Sample volume refers to the entire contents of any well, including bufferblank, or any combination of master mix and nucleic acids.IMPORTANT! All wells on one plate must contain the same reaction volume.6. Choose from the following options:If performing an…absolute or relativequantification run onlyabsolute quantification runwith a dissociation curveThen…go to “Step 6 – Saving the Plate Document as aTemplate” on page 3-24.add a temperature ramp to generate dissociationcurve data as explained on page 3-23.3-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 5 – Programming the Plate Document MethodProgramming aTemperatureRamp forDissociationCurve AnalysisTo generate the data required to perform a dissociation curve analysis, the 7900HTinstrument must be programmed to run a ‘temperature ramp’ in which it slowlyelevates the temperature of the samples while collecting fluorescence measurementsonce every 7-10 seconds (see page 6-38 for a detailed explanation).1. In the Instrument tab of the plate document, select the Thermal Profile tab.2. Click the step to the left of the location you want to place the new stage.3. In the Thermal Profile tab, click .The SDS software inserts a temperature ramp at the end of the thermal profileconsisting of a set of default steps.4. You can customize the default temperature ramp, however Applied Biosystemsrecommends that you observe the following guidelines to ensure the maximumresolution for the run (the greatest separation of the derivative peaks during theanalysis).GuidelineExampleThe Start and End steps ofthe temperature ramp must:• be separated by aminimum temperaturedifference of 35 °C.• elapse 15 seconds (0:15)each.End stepStart stepThe ramp rate setting for theEnd step of the temperatureramp must be 2 %.IMPORTANT! You mustdeselect the 9600 Emulationcheck box to modify theRamp Rate Settings.End stepramp ratesettingThe temperature ramp in thethermal profile must containa data collection icon.Datacollectionicon5. Go to “Step 6 – Saving the Plate Document as a Template” on page 3-24.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-23DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunStep 6 – Saving the Plate Document as a TemplateUser AccessRequirementAdjusting theDisplay Settings(Optional)If using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save a plate document as a plate document template.Because plate documents created from the plate document template will retain itsdisplay settings, configure the display settings of the plate document template as youwould like the child plate documents to be displayed.1. Click (select View > Display Setting).2. In the Display Settings dialog box, configure the display settings forthe Results Grid, and the Results Table.For more information on the Display Settings dialog box or to view theprocedures for configuring the display settings for the plate document template,click to open the Sequence Detection Systems Software Online Help.3. When finished, click .The SDS software applies the new display settings to the plate document.4. Go to “Saving the Plate Document as a Template (Optional)” on page 3-25.3-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 6 – Saving the Plate Document as a TemplateSaving the PlateDocument as aTemplate(Optional)IMPORTANT! Saving the plate document as a plate document template is an optionalstep and recommended for instances where the document can be used to createduplicate plate documents for a series of plates with identical assay configurations. Ifyou choose not to use your plate document as a plate document template, go to“Configuring the Plate Document Information (Optional)” on page 3-28.To save the plate document as an SDS 7900HT Template Document:1. Select File > Save As.2. In the Save in field of the Save As dialog box, navigate to theAppliedBiosystems > SDS2.2.1 > Templates directory.Note: By saving the file to the Templates directory, it becomes available fromthe Template drop-down list in the New Document dialog box.3. Select File of type > SDS 7900HT Template Document (*.sdt).4. Click the File name field, and enter a name for the plate document template.5. Click .The software saves the plate document template.6. If the software displays one or more warnings, read the note and click .7. Select File > Close.If the software prompts you to save the plate document, click .The SDS software closes the plate document template.8. Create a plate document from the plate document template as explained onpage 3-26.To save the plate document template to the SDS Enterprise Database:1. Select File > Save Template to Database.2. In the Save Document as Template dialog box, click .3. If the software displays one or more warnings, read the note and click .4. In the Template Name field of the Save Template to Database dialog box, entera name for the plate document template (up to 128-characters), and click.5. Select File > Close.If the software prompts you to save the plate document, click .The SDS software closes the plate document template.6. Create a plate document from the plate document template as explained onpage 3-26.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-25DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunStep 7 – Creating a Plate Document from the TemplateUser AccessRequirementOptions forCreating PlateDocuments fromthe TemplateIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to create a plate document from a plate documenttemplate.The SDS software offers two options for creating plate documents from a platedocument template: individually or in batches.Option Description See PageCreate an individual platedocument from the platedocument templateCreate multiple plate documentsusing the Template Batch utilityIMPORTANT! If using theSDS Enterprise Database, youmust choose this option.The procedure below explains how tocreate a single plate document from aplate document template for running aplate. By repeating the procedure, youcan create as many plate documents asneeded.As a faster alternative to the optionabove, the software includes aTemplate Batch utility that cansimultaneously create multiple platedocuments from the plate documenttemplate.Follow theprocedurebelow.4-36Creating a SDSPlate Documentfrom a Template1. In the SDS software, click (or select File > New).2. Configure the New Document dialog box:• Assay drop-down list – Select the same assay as the plate documenttemplate.• Container drop-down list – Select the same plate type as the platedocument template.• Template drop-down list – Select the plate document template (*.sdt)created on page 3-24.If the plate document template does not appear inside the Template drop-downlist, select the file as explained below:a. Click .b. In the Look in field of the Open dialog box, navigate to and select the platedocument template (*.sdt) created on page 3-24.c. Click .The Template drop-down list displays the plate document template.Note: The Template drop-down list displays all plate document templatescontained in the Templates subdirectory of the SDS program directory.• Barcode field – (Optional) Do one of the following:– Enter the bar code number for the plate, or– Scan the plate bar code using the hand-held bar code scanner.3. Click to create a new plate document from the plate document template.3-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 7 – Creating a Plate Document from the Template4. Configure the new plate document with sample names and plate information asexplained on page 3-28.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-27DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a RunStep 8 – Applying Sample and Plate InformationUser AccessRequirementApplying SampleNames to thePlate DocumentIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to apply (or modify) sample names to the plate document.The plate document must contain sample attributes to effectively organize andanalyze data produced from the run. Once applied, the software displays the samplenames in the plate grid and table views.Note: You can apply sample names after the plate has been run, but they must beadded before the analysis of the run data.Note: The SDS software provides the ability to import setup table information(detector, detector task, and sample name layouts) into a plate document from atab-delimited text file. See “Importing Plate Document Setup Table Files” onpage A-2 for more information.1. In the plate grid, select the wells containing the first sample.2. Click the Sample Name field, enter a name for the sample, and press Enter.The software labels the selected wells with the new sample name.3. Repeat steps 1 through 2 for all remaining samples.Sample nameappears hereSample Name field4. Configure the plate document with plate information as explained below.Configuring thePlate DocumentInformation(Optional)1. In the SDS software, select Tools > Document Information.2. In the Document Information dialog box, edit the Barcode, Operator, orPlate Comments information.IMPORTANT! If using an SDS Enterprise Database:• The bar code entered into the plate document must be unique and cannot beused in another plate document.• The software automatically populates the Operator field with the name of theuser who logged into the SDS software and performed the data collection.The software populates the field only after the plate document has been run.• Do not modify the bar code of a plate document that contains data from a run.3. When finished, click .4. Run the plate document and associated plate as explained on page 3-29.3-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Step 9 – Running the Plate on the 7900HT InstrumentStep 9 – Running the Plate on the 7900HT InstrumentUser AccessRequirementOptionsfor RunningSDS PlatesThere is no access requirement. All users can run plates that have been saved to theSDS Enterprise Database.IMPORTANT! A plate document must be saved to the database before a userbelonging to the Operator User Group can run it.The 7900HT instrument can run prepared plates individually or in groups using theZymark Twister Microplate Handler.IMPORTANT! If you are not using a Zymark Twister Microplate Handler, you mustrun plates individually.Choose one of the following options to run the plate:To run the plate… Description See Pageindividuallyas part of a group(Automated Operation)1. Prepare the consumable (optical plate orTaqMan ® Low Density Array)2. Run the plate/array individually using the SDSsoftware.1. Prepare the consumables (optical plates orTaqMan ® Low Density Arrays)2. Run the plate/array with others as part of a batchfrom the Automation Controller Software usingthe Zymark Twister Microplate Handler.IMPORTANT! You must have a Zymark TwisterMicroplate Handler to run plates using this option.4-54-234-54-31Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-29DRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Chapter 3Preparing a Run3-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Run.fm

Operating the Instrument 44In This Chapter Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4Section 4.1 Consumable Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8Preparing TaqMan Low Density Arrays for Use. . . . . . . . . . . . . . . . . . . . . . . . . . 4-10Section 4.2 Running an Individual Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-23Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . 4-25After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29Section 4.3 Automated Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-31Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34Running Plates Using the Automation Controller Software . . . . . . . . . . . . . . . . . 4-41After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-1DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when creating andmodifying plate documents as described in this chapter. This section describes thetypes of actions you may need to perform depending on how your administrator hasconfigured the database. For more information on any of the features describedbelow, see the SDS Enterprise Database for the Applied Biosystems 7900HT FastReal-Time PCR System Administrators Guide.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.When the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 4-1Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.When the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.4-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Notes for Database UsersDescription of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 4-2Electronic Signature Verification Dialog Box OptionsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-3DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentBefore You BeginPre-RunChecklistThe following tasks must be complete to run plates on the 7900HT instrument. Seethe associated page number for details on each procedure.Done Check See PageA background run has been performed in the last month 7-16A pure dye run has been performed in the last 6 months 7-20The instrument tray does not contain a plateIMPORTANT! The instrument tray must be empty to begin a run. Ifthe instrument tray contains a plate, you must eject and remove it.4-44If using an Automation Accessory, also check the following…The output stack does not contain plates. –WorkflowOverview1. Prepare your experiments for use on the 7900HT instrument:– Prepare optical plate(s) (see page 4-8).– Prepare TaqMan ® Low Density Array(s) (see page 4-10).2. Prepare and run the 7900HT instrument:– Stand-alone Operation (see page 4-23).– Automated Operation using the SDS Enterprise Database (see page 4-32).– Automated Operation without the database (see page 4-34).3. Analyze the run data.– Allelic Discrimination Analysis (see page 5-5).– Absolute Quantification Analysis (see page 6-5).– Relative Quantification Analysis (see page 6-15).– Dissociation Curve Analysis (see page 6-37).4-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Section 4.1 Consumable PreparationSection 4.1Consumable PreparationIn This Section Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Compatible Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6Preparing Optical Plates for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8Preparing TaqMan Low Density Arrays for Use. . . . . . . . . . . . . . . . . . . . . . . . . . 4-10Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-5DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentPreventing ContaminationContaminationand the5´ NucleaseAssayGeneral PCRPracticesPCR using the 5´ nuclease assay requires special laboratory practices to avoid falsepositive amplifications (Kwok and Higuchi, 1989). The assay’s logarithmicamplifications can potentially amplify a single DNA molecule (Saiki et al., 1985;Mullis and Faloona, 1987).Observe the following guidelines when assembling optical plates or loadingTaqMan ® Low Density Arrays for use on the 7900HT instrument:• Wear a clean lab coat (not previously worn while handling amplified PCR productsor used during sample preparation) and clean gloves when preparing samples forPCR amplification.• Change gloves whenever you suspect they are contaminated.• Maintain separate areas, dedicated equipment, and supplies for:– Sample preparation– PCR setup– PCR amplification– Analysis of PCR products• Never bring amplified PCR products into the PCR setup area.• Open and close all sample tubes carefully. Try not to splash or spray PCR samples.• Keep reactions and components capped as much as possible.• Use positive-displacement pipets or aerosol-resistant pipet tips.• Clean lab benches and equipment with 10% bleach solution.Compatible ConsumablesConsumables forUse withthe 7900HTInstrumentTable 4-1 Compatible ConsumablesConsumable TypeUse these…Compatible SealsNot these…ABI PRISM 384-Well OpticalReaction Plates• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)384-Well Septa4-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Compatible ConsumablesTable 4-1Compatible ConsumablesConsumable TypeUse these…Compatible SealsNot these…ABI PRISM 96-WellOptical ReactionPlatesOptical 96-WellFast ThermalCycling PlatesTaqMan ® LowDensity Arrays• Compression Pads:– Standard pad for manual operation– ABI PRISM Snap-On CompressionPad (reusable)• 96-Well Plate Seals:– ABI PRISM Optical Adhesive Covers(quantity 100)– Optical Adhesive Covers(quantity 25)– ABI PRISM Optical Caps(flat caps only)• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)None (self sealing)• ABI PRISM Optical Caps(round caps)• 96-Well SeptaNone (self sealing)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-7DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentPreparing Optical Plates for UsePreparing Platesfor Use on the7900HTInstrument1. Prepare the reactions in an optical plate by aliquoting reagents, enzyme, andsamples to the appropriate wells of an optical plate.IMPORTANT! The arrangement of the reactions (samples and assays) on theplate must match the configuration of information (sample names anddetectors/markers) on the corresponding plate document.2. Seal the optical plate with a seal that is compatible with the 7900HT instrument(Figures 4-3 and 4-4 on page 4-9).Note: See “Compatible Consumables” on page 4-6 for a list of seals for usewith the Applied Biosystems 7900HT Fast Real-Time PCR System.3. Briefly centrifuge the plate to collect the reactions at the bottom of the wells andto eliminate any air bubbles that may be present.4. Visually verify that each reaction is positioned at the bottom of its well.Correct PositionIncorrect PositionThe sample is positionedcorrectly in the bottom ofthe well.The sample lies on theside wall because theplate was not centrifuged.An air bubble lies at thebottom of the wellbecause the plate wasnot:• Centrifuged with enough force, or• Centrifuged for enough time5. If running an ABI PRISM 96-Well Optical Reaction Plate, apply the appropriatecompression pad to the sealed optical plate.If you are going to run the plate:• Individually using the SDS software, then apply a standard compressionpad (Figure 4-3)• As part of a batch using the Automation Controller Software, then apply anABI PRISM Snap-On Compression Pad (Figure 4-3)IMPORTANT! The ABI PRISM Snap-On Compression Pad/plate assemblymay still be hot after the Zymark ® Twister Microplate Handler loads it intothe Plate Handler’s output stack. Please wait at least 30 seconds beforemanually handling the Snap-On Compression Pad/plate assembly.6. Choose from the following. If performing:– Absolute or Relative Quantification, or Dissociation Curve AnalysisRun the plate as explained in “Running the Plate” on page 4-25.– Allelic Discrimination4-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing Optical Plates for Usea. Load the plate onto a designated thermal cycler and perform the PCR.b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells andto eliminate any air bubbles that may have formed during thermal cycling.c. Run the plate as explained in “Running the Plate” on page 4-25.‘Snap-On’Compression Pad(for automated runs)Standard compressionpad (for single-plateruns)Optical adhesivecoverOROptical Caps(flat caps only)Figure 4-396-Well Optical Reaction Plate AssemblyOptical adhesivecover384-wellplateFigure 4-4384-Well Optical Reaction Plate AssemblyApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-9DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentPreparing TaqMan Low Density Arrays for UseAbout the Low Density ArraysThe TaqMan Low Density Arrays are research tools for profiling gene expressionusing the Comparative C T Method of relative quantification. The Low Density Arrayevaluates from one to eight cDNA samples or controls generated from human totalRNA in a two-step RT-PCR experiment.How It WorksLow DensityArrayComponentsThe Low Density Array functions as an array of reaction vessels for thePCR/sequence detection step. Typically, the wells of the Low Density Array containApplied Biosystems fluorogenic 5´ nuclease assays (TaqMan ® reagents; see the notebelow) that detect the real-time amplification of user-specified targets. Relativelevels of gene expression are determined from the fluorescence data generated duringPCR using the Applied Biosystems 7900HT Fast Real-Time PCR System RelativeQuantification software.Note: In this document, the term TaqMan reagents refers to 5´ exonuclease assayreagents that feature Applied Biosystems TaqMan ® primers and probes (includesApplied Biosystems PDARs, TaqMan ® SNP Genotyping and Gene ExpressionAssays, Custom TaqMan ® SNP Genotyping and Gene Expression Assays, and anycustom TaqMan reagents.)The Low Density Array (Figure 4-5) acts as a vessel for real-time PCR. It consists ofa series of 384 interconnected wells divided into eight sets of assays. Each setprovides a user-specified number of replicates. Each well contains dried AppliedBiosystems TaqMan primers and probes for one mRNA target.Fill reservoir (1 of 8)A reservoir for the cDNA sample orcontrol before it is centrifugallyloaded into the wellsFill consumableThe segment of the Low DensityArray that contains the eight fillreservoirs. (After the Low DensityArray is centrifuged and sealed, thefill consumable is trimmed off.)GR2156aBarcode Provides coded access toLow Density Array configurationdatabasesFigure 4-5The TaqMan Low Density Array4-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for UseInternal StructureFigure 4-6 illustrates the Low Density Array internal structure.Fill portVent portFill reservoirMain channelFeeder channelReaction wellFigure 4-6TaqMan Low Density Array, Internal StructureDetectorsGenomic DNAContaminationThe Low Density Array permits the amplification of target and an externalendogenous control cDNA using fluorogenic 5´ nuclease assays. The assays consistof two reactions, each a complete PCR system with corresponding probe andprimers.The fluorogenic probes of the assays function as follows:• Probes labeled with the FAM dye detect the amplification of user-specifiedcDNA targets.• Probes labeled with the FAM dye detect the amplification of the externalendogenous control.TaqMan probes and primers for the cDNA targets span exon-exon junctions tominimize the contribution of contaminating genomic DNA. Performance testsdemonstrate that 5´ exonuclease assays can be run with samples containing up to10,000 copies of genomic DNA without detection of contaminants, unless otherwisespecified by Applied Biosystems.External Endogenous Control AssayNote that the commonly-used external endogenous control assay is not RNA-specificand consequently is affected by genomic DNA contamination. However, because ofthe extremely high expression level of rRNA, even gross DNA contamination has anegligible effect on the relative quantification values obtained from the Low DensityArray.Single-PlexReactionsQuality ControlThe Low Density Array is recommended for use with single-plex TaqMan reagents(for example, FAM dyes). An external endogenous control assay (for example,FAM-18S or FAM-GAPDH) is used as a calibrator in relative quantificationcalculations.Note: For more information on relative quantification, see ABI PRISM ® 7700Sequence Detection System User Bulletin #2: Relative Quantitation of GeneExpression (PN 4303859).Functional verification of the preloaded probes and primers inside the Low DensityArray is performed as part of the Applied Biosystems manufacturing quality controlprocess.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-11DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentAbout the Centrifuge SystemAfter the fill reservoirs of a Low Density Array have been loaded with the cDNAsample or control, the centrifuge system is required to distribute the cDNA sample orcontrol to the reaction wells. The centrifuge system supports the simultaneousloading of up to 12 Low Density Arrays.RequiredCentrifuge andAccessoriesTo properly centrifuge the Low Density Arrays, you will need the followingcentrifuge and centrifuge accessories:• Sorvall ® Legend T CentrifugeGR2170Figure 4-7Sorvall Legend T Centrifuge• Sorvall ® /Heraeus “Tool-less” 750-mL Swing-Out Rotor Body• A set of Sorvall ® /Heraeus Custom Buckets (four), each with its own card holder(Contact your Applied Biosystems representative to order the custom buckets.)• Current firmwareIf you are upgrading an existing Sorvall Legend T or Legend RT Centrifuge,you may also need to upgrade the firmware so that it can recognize the LowDensity Array bucket type.CentrifugeSystemComponentsFigure 4-8 illustrates the centrifuge system components.Centrifuge rotorProvides the rotational drive fora set of four centrifuge bucketsCard holderSupports the Low DensityArray’s fill reservoirs duringcentrifugationGR2152GR2153GR2154BucketContains the Low DensityArrays and card holderduring centrifugationFigure 4-8Centrifuge System Components4-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for UseLaboratory SetupSetting theBucket Type forthe Low DensityArray.The centrifuge system is designed to be placed on a standard laboratory bench. Itrequires access to 115 VAC, 60 Hz electrical power. Consult the owner’s manualshould for specific operating and site preparation instructions.1. Power on the centrifuge.2. Following the instructions in the Sorvall Legend T Centrifuge operator’smanual, use the front panel controls on the centrifuge to set the bucket type forthe centrifuge to 15679Bucket type controlScrolling controlIMPORTANT! Be sure to set the correct bucket type. Setting the correct buckettype ensures that the maximum rotational speed stays within the manufacturer’sspecified limits.3. Using the front panel controls, set the following operational parameters:ParameterValueUp ramp rate 9Down ramp rate 9Rotational speed 1200 rpm (331 × g)Centrifugation time1 minrpm indicatorButtons 1–4 areprogrammingbuttons.Up ramprate controlDown ramprate controlrpmcontrolsTimecontrols4. Power off the centrifuge.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-13DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentAbout the SealerThe microfluidic card sealer is required to isolate the wells of the Low Density Arrayafter it is loaded with cDNA samples or controls and master mix. The sealer uses aprecision stylus assembly (carriage) to seal the main fluid distribution channels of theLow Density Array.SealerComponentsHandleUsed to move the carriage,which contains the stylusassemblyLow Density ArrayShown foil-side up, with fill reservoirs on the right(carriage moves from left to right during sealing, inthe direction of the arrows etched into the base)Insert plateHolds the Low DensityArray during sealingGR2172BaseProvides a stable platform forthe Low Density Array and atrack for the stylus assemblyFigure 4-9Sealer Components (Shown with Cover Removed)Laboratory SetupThe microfluidic card sealer is designed to be placed on a standard laboratory bench.It requires no electrical power.Loading the TaqMan Low Density ArraysEquipment andMaterials NeededGuidelines forLoading theCardsYou need the following equipment and materials to load the sample-specific PCRreaction mix into the fill reservoirs of the Low Density Arrays:• Rainin F-100 micropipette (100-µL)• Rainin Fine Point pipet tips (100-µL)• TaqMan Low Density Array(s)• Sample-specific PCR reaction mix (see the protocol accompanying your LowDensity Array chemistry for instructions on preparing the reaction mix)Follow the guidelines below to ensure proper loading of the Low Density Arrays.• Do not remove a Low Density Array from its packaging until the packaging hasreached room temperature and you are ready to load it with the sample-specificPCR reaction mix.IMPORTANT! Prolonged exposure to indoor lighting can photo-degrade thefluorescent probes contained inside the Low Density Array. Do not expose theLow Density Array to sunlight.• Do not twist or bend the fill consumable portion of the Low Density Array(Figure 4-5 on page 4-10) before loading.IMPORTANT! To successfully load the sample-specific PCR reaction mix, it iscritical that the main channels leading from the Low Density Array’s fillreservoirs to its wells are open and undamaged.4-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for UseLoading the FillReservoirs withSample-SpecificPCR ReactionMix1. When the packaging has reached room temperature and you are ready to loadPCR reaction mix, carefully remove a Low Density Array from its packaging.2. Place the Low Density Array on a lab bench, with the foil side down.3. Load 100 µL of the desired sample-specific PCR reaction mix into a 100-µLmicropipette.4. Hold the micropipette in an angled position and place the tip in the fill port.Note: There is a fill port on the left arm of each fill reservoir; it is the larger ofthe two holes.Fill portVent portGR2158IMPORTANT! Do not allow the tip to contact and possibly damage the coatedfoil beneath the fill port.5. Dispense the sample-specific PCR reaction mix so that it sweeps in and aroundthe fill reservoir toward the vent port.IMPORTANT! Pipette the entire 100 µL into the fill reservoir.IMPORTANT! Do not allow the tip to contact and possibly damage the coatedfoil beneath the fill port.IMPORTANT! Be careful when pushing the micropipette plunger to its secondstop position (to expel the sample-specific PCR reaction mix from the tip). If alarge amount of air is released, it can push the reaction mix out of the fillreservoir via the vent port.GR21596. To transfer the sample-specific PCR reaction mix from the fill reservoirs intothe reaction wells, the Low Density Array must be centrifuged.Continue with “Centrifuging the TaqMan Low Density Arrays” on page 4-16.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-15DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentCentrifuging the TaqMan Low Density ArraysEquipment andMaterials NeededPlacing Cards inthe BucketsYou need the following equipment and materials to centrifuge the Low DensityArrays:• TaqMan Low Density Array(s), containing sample-specific PCR reaction mix(page 4-14)• Sorvall ® Legend T Centrifuge• Sorvall ® /Heraeus Custom Buckets (four required), each with its own card holder• Blank balance cardsNote: The centrifuge holds four Sorvall/Heraeus buckets. Each bucket holds up tothree Low Density Arrays (loaded and/or blank balance cards).1. Obtain an empty Sorvall/Heraeus Custom Bucket and a card holder for thecentrifuge.IMPORTANT! The Sorvall/Heraeus buckets and card holders required for theLow Density Arrays are custom-made. Do not use any other bucket/card holdersystem for this procedure.2. Place the bucket on a lab bench, with the label facing you.3. Insert Low Density Arrays into the card holder, making sure that:• The fill reservoirs project upwards out of the card holder• The reaction wells face the same direction as the “This Side Out” labelIMPORTANT! Use blank balance cards to fill any remaining positions in theracks.Note: The card holder supports the Low Density Array fill reservoirs duringcentrifugation.GR21554. Place a filled card holder in the bucket so that the “This Side Out” label facesthe front of the bucket, which has the Sorvall emblem on it.Sorvallemblem hereGR21604-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for UsePlacing theBuckets in theCentrifuge1. Make sure the centrifuge is powered on, but is not rotating.2. Open the centrifuge cover by pressing the OPEN button.OPEN cover button3. Place a loaded bucket onto an open rotor arm of the centrifuge.Note: Make sure the bucket can swing easily within its slotted position on therotor arm.4. Place the remaining buckets onto the rotor arms, per step 3.The manufacturer recommends running the centrifuge withall four buckets, even if only two buckets contain Low Density Arrays.Make sure the buckets and their contents are balanced.Opposing buckets should have matching weights. If the buckets are not fullyloaded with Low Density Arrays containing the sample-specific PCR reactionmix, place blank balance cards and card holders into the buckets.GR21615. Close the centrifuge cover.Centrifugingthe Cards1. Press the START button.STARTbuttonThe centrifuge starts, then automatically stops after 1 min, per the programmedsequence.2. Repeat step 1 so that the Low Density Arrays are centrifuged for a total of twoconsecutive, 1-min spins to ensure complete distribution of the sample-specificPCR reaction mix.3. Open the centrifuge cover by pressing the OPEN button.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-17DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the Instrument4. When the cover has fully opened, remove the buckets from the centrifuge, thenremove the card holders from the buckets.5. Remove all Low Density Arrays from the buckets by gently lifting them by theircarrier sides.6. Examine the Low Density Arrays to determine whether filling is complete.The amount of cDNA sample or control remaining in the fill reservoirs shouldbe uniform and consistent from reservoir to reservoir.• If there is excess cDNA sample or control remaining in a fill reservoir (asshown below), filling is incomplete. Proceed to the next step.• If a fill reservoir is completely drained (as shown below), it is possible thatsome wells were not filled properly.– The Low Density Array should not be processed further. Use anotherLow Density Array.– Alternatively, you can process the Low Density Array further; however,you should void the results for the affected fill reservoir (sample).7. Evaluate the filling as follows:If all fill reservoirs are...not uniform after the firstcentrifuge cyclenot uniform after theadditional centrifuge cycleThen...the Low Density Array may be centrifuged again in thesame manner as before for 1 additional min.• The Low Density Array should not be processedfurther. Use another Low Density Array.• Alternatively, you can process the Low Density Arrayfurther; however, you should void the results for theaffected fill reservoir (sample).IMPORTANT! Do not exceed 1200 rpm or accumulated centrifugation times ofmore than 3 min. Excessive centrifugation speeds and times may deform theLow Density Array.4-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for UseSealing the TaqMan Low Density ArraysEquipment andMaterials NeededGuidelines forSealing the CardsPositioning theSealerYou need the following equipment and materials to seal the Low Density Arrays:• Microfluidic card sealer• Sturdy lab bench• TaqMan Low Density Array(s), containing sample-specific PCR reaction mixand centrifuged (page 4-16)• ScissorsFollow the guidelines below to ensure the Low Density Arrays are properly sealed.• Seal the Low Density Arrays as soon as possible following centrifugation. Therisk of cross-contamination is minimized by the sealing process.• Always place the Low Density Arrays into the sealer so that the sealer’s carriage(stylus assembly) travels toward the fill reservoir end of the Low Density Array.• Always seal in the direction indicated by the arrows etched into the base of thesealer to ensure that the cDNA sample or control is pushed from the LowDensity Array’s channels into its fill reservoirs.The sealer is best operated by pushing the carriage away from you in a near-to-farmotion (not in a horizontal, side-to-side motion). Follow the procedure below tocorrectly position the sealer.1. Place the sealer on a sturdy lab bench, approximately waist high so that it can beeasily used.2. Turn the sealer so that the front end (where sealing starts) is closest to you andthe back end is farthest from you.Note: In the correct position, the arrows on the sealer are pointing away fromyou.CarriageGR2171StartingpositionInserting a CardInto the Sealer1. Place the sealer’s carriage in its starting position.IMPORTANT! Never insert a Low Density Array into the sealer if the carriage isnot in its starting position. If you move the carriage across the Low DensityArray to return it to its starting position, serious damage to the Low DensityArray will occur.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-19DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the Instrument2. Insert a Low Density Array into the sealer:a. Orient the Low Density Array in the proper direction over the sealer’s insertplate. The Low Density Array’s fill reservoir end should be the end closestto the arrows etched in the base of the sealer.b. Insert the Low Density Array, foil side up, on top of the insert plate.GR21773. Gently push the Low Density Array until it is seated securely in the insert plate.Note: Two metal pins on the sealer’s insert plate project into correspondingholes molded into the Low Density Array’s carrier to provide mechanicalalignment for the sealing process. When properly seated, the Low DensityArray’s foil surface should be level with the base of the sealer.Note: Four spring clips ensure that the Low Density Array is held in the properposition.PinPinGR2174Sealing a Card1. Push the carriage across the base of the sealer in the direction of the arrows. Useone firm, smooth motion until you reach the end of the sealer.The sealer has mechanical stops at both ends to prevent thecarriage from coming off. Do not use excessive force or speed when pushing thecarriage. Using force will not increase the sealing quality but only cause wear tothe sealer.End positionGR2176Move in thisdirection:4-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Preparing TaqMan Low Density Arrays for Use2. Remove the sealed Low Density Array by grasping its sides and lifting it off thesealer’s insert plate.Note: In the middle of the sealer’s insert plate, there is a thumb slot to help youeasily access one side of the Low Density Array.Thumb slotGR21753. Inspect the Low Density Array for proper sealing. The indentations from thestylus assembly should match up with the Low Density Array’s main channels.If the indentations do not match up or if the foil is in any way damaged, do notuse the Low Density Array.4. Return the carriage to its starting position on the base of the sealer.IMPORTANT! Do not move the carriage back before removing the Low DensityArray.Trimming Off theFill Reservoirs1. Using scissors, trim the fill reservoirs from the Low Density Array. Use the edgeof the Low Density Array’s carrier as a guide.PHYSICAL INJURY HAZARD. Take care when trimmingoff the fill reservoirs. Use scissors rather than razor blades or other unprotectedcutting devices.GR2168The Low Density Array is now ready to be run on the 7900HT instrument.2. Choose from the following. If performing:– Absolute or Relative Quantification, or Dissociation Curve AnalysisRun the plate as explained in “Running the Plate” on page 4-25.– Allelic Discriminationa. Load the plate onto a designated thermal cycler and perform the PCR.b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells andto eliminate any air bubbles that may have formed during thermal cycling.c. Run the plate as explained in “Running the Plate” on page 4-25.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-21DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the Instrument4-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Section 4.2 Running an Individual PlateSection 4.2Running an Individual PlateIn This Section Saving the Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-24Running a Single Plate (Using the SDS Software) . . . . . . . . . . . . . . . . . . . . . . . . 4-25After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-29Pre-RunChecklistThe following tasks must be complete to run plates on the 7900HT instrument. Seethe associated page number for details on each procedure.Done Check See PageA background run has been performed in the last week 7-16A pure dye run has been performed in the last 6 months 7-20The instrument tray does not contain a plateIMPORTANT! The instrument tray must be empty to begin a run. Ifthe instrument tray contains a plate, you must eject and remove it.4-44WorkflowOverview1. Save the SDS Plate Document (see page 4-24).2. Load and run the prepared optical plate or Low Density Array (see page 4-25).3. Analyze the run data.– Allelic Discrimination Analysis (see page 5-5).– Absolute Quantification Analysis (see page 6-5).– Relative Quantification Analysis (see page 6-15).– Dissociation Curve Analysis (see page 6-37).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-23DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentSaving the Plate DocumentUser AccessRequirementSaving the PlateDocument forSingle PlateOperationIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save a plate document to the database.Before the plate document can be run, you must save it to the SDS EnterpriseDatabase or as an SDS 7900HT Document (*.sds).To save as an SDS 7900HT Document:1. In the SDS software, select File > Save As.2. In the Look in field of the Save As dialog box, navigate to and select a directoryfor the software to receive the new file.3. In the File name field either:• Enter a file name for the plate document file, or• Enter or scan the bar code number for the plate into the field.Note: The SDS software does not require that the file name match the bar codeof the corresponding plate.4. Click .The software saves the plate document to the specified directory.5. Run the plate document and associated plate as explained on page 4-25.To save to the SDS Enterprise Database:1. Select File > Save Document to Database.2. Configure the Save Document to Database dialog box:a. In the Comment field, enter a brief description of the plate document(up to 255-characters).If saving a plate document file to the database, you will loose the file nameunless you enter it into the Comments field.b. Click .3. In the Saved Document dialog box, click .4. Run the plate document and associated plate as explained on page 4-25.4-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Running a Single Plate (Using the SDS Software)Running a Single Plate (Using the SDS Software)User AccessRequirementRunning the PlateThere is no access requirement. All users can run plates that have been saved to theSDS Enterprise Database.1. In the SDS software, select the Instrument tab of the plate document.2. In the Real-Time or Plate-Read tab of the Instrument tab, click .The instrument tray rotates to the OUT position.3. Place the prepared optical plate or Low Density Array into the instrument trayas shown below.Before loading the plate or Low Density Array onto the instrument tray, makesure that:• The associated plate document is open in the SDS software.• The optical plate or Low Density Array has been sealed.Well A1Notched cornerBar codeWell A1Notched cornerBar codeIMPORTANT! The A1 position is located in the top-left side of the instrument.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-25DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the Instrument4. Do one of the following:• If performing a real-time run, click .• If performing an end-point run, click .The instrument tray rotates to the IN position and the instrument performs therun or plate-read.Note: Before starting the run, the instrument may pause (up to 15 minutes) toheat the heated cover to the appropriate temperature.Note: For more information on the elements of the Real-Time and Plate-Readtabs, click and see the Sequence Detection Systems Software Online Help.The following options are available during and after the completion of the run:To…See PageMonitor the progress of the run 4-27Stop the runIMPORTANT! If you must stop a run in-progress for any reason,carefully read the instructions on page 4-28 before halting the run.4-28Open the instrument tray (after the run) 4-29Analyze the run data after the run is complete 4-294-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Running a Single Plate (Using the SDS Software)MonitoringInstrumentProgressThe SDS software displays instrument status and run progress in the Real-Time tab(real-time runs) or the Plate Read tab (end-point runs) of the respective platedocument. Figure 4-10 shows examples of the tabs during operation of the 7900HTinstrument.Real-Time Tab(Real-Time Runs)Plate Read Tab(End-Point Runs)Figure 4-10Real-Time and Plate Read Tabs of the SDS Software• Status – Displays the condition of the 7900HT instrument• Time Remaining – Displays the calculated time remaining in the runTemperature group box (Real-Time Plate Documents Only)• Block – Displays the actual temperature of the sample block module• Cover – Displays the actual temperature of the heated cover• Sample – Displays the calculated temperature of the samplesCycle group box (Real-Time Plate Documents Only)• Rep – Displays the current cycle repetition• Stage – Displays the current stage of the thermal cycling• State – Displays the current condition of the cycle stage• Step – Displays the current step being run• Time – Displays the calculated time remaining in the current stepData Collection Stamp group box (End-Point Plate Documents Only)• Pre – Displays the date that the instrument performed the pre-read.• Post – Displays the date that the instrument performed the post-read.Note: A ‘pre’ read is a plate read performed before a plate has undergone thermalcycling.Note: A ‘post’ read is a plate read performed after a plate has undergone thermalcycling.Note: For more information on the elements of the Real-Time and Plate-Read tabs,click and see the Sequence Detection Systems Software Online Help.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-27DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentStopping theRun Using theSDS SoftwareIMPORTANT! Read the following directions carefully before stopping arun-in-progress.Stopping End-Point Runs (Allelic Discrimination)In the Plate-Read tab of the plate document, click .Stopping Real-Time Runs (Absolute or Relative Quantification)Has the Cover reading in the Real-Time tab reached 104 °C?• No (the heated cover temperature is below 104 °C)In the Real-Time tab of the plate document, click .• Yes (the heated cover temperature has reached 104 °C)The instrument is running the plate and has begun thermal cycling. Determine acourse of action from the options in the following table:Reason for Stopping the RunForgot to make a change to the platedocument such as adding a detector,detector task, or sample name(not including mistakes to thetemperature profile)• Forgot to add a reagent to the plate(such as enzyme or master mix)• Programmed the plate documentwith the wrong thermal profileThen…do not stop the run.Allow the instrument complete the run,then edit the plate document beforeanalyzing the data. The software does notuse detector or sample information untilyou analyze the plate document.1. In the Real-Time tab of the platedocument, click .2. Determine how far into the run theinstrument has progressed.3. Based on the state of the run, determinewhether the plate can be re-run.4. If necessary, eject the plate and add themissing reaction component.5. If desired, re-run the plate by re-creatingthe plate document.4-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

After the RunAfter the RunUser AccessRequirementEjecting a Plate(Opening andClosing theInstrument Tray)If using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save a plate document.View the instrument status icon to determine the plate document to use to eject the plate.Instrument StatusConnected toplatenameAction1. In the SDS software, select Window >(the plate documentconnected to the instrument).2. In the plate document, select the Instrument tab3. Select the Plate-Read or Real-Time tab.4. Click .Disconnected1. In the SDS software, click (or select File > New).2. Click .3. Select the Instrument tab.4. Select the Plate-Read or Real-Time tab.5. Click .Saving theRun DataAfter the run, you must either save the results to the SDS Enterprise Database or savethe run data to the SDS 7900HT Document (*.sds).To save the run data to the SDS 7900HT Document:1. In the SDS software, select File > Save.To save the run data to the SDS Enterprise Database:IMPORTANT! If you saved the plate document to the database prior to running theplate, the SDS software stored the run data as a temporary file in the /temp directory. Ifyou closed your plate document at the end of the run without saving it, open the tempdirectory and rename the temporary file to *.sds and you will be able to open the file.1. Select File > Save Document to Database:2. In the Comment field of the Save Document to Database dialog box, enter abrief description of the plate document (up to 255-characters).Note: If saving a plate document file to the database, you will loose the filename unless you enter it into the Comments field.3. Click .4. In the Saved Document dialog box, click .Analyzing theRun DataSection 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-29DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentDisconnectingthe Software fromthe InstrumentThe SDS software has the ability to halt all communications with the 7900HTinstrument. The ‘disconnect’ option permits the simultaneous operation of both theSDS software and the Automation Controller Software. Because both programscontrol the Applied Biosystems 7900HT Fast Real-Time PCR System, one programmust relinquish control of the 7900HT instrument before the other can be used tooperate it.To…disconnect thesoftware fromthe instrumentreconnect the software(once disconnected)Then…1. In the SDS software, select the Instrument tab of the openplate document.2. Select the Real-Time or Plate-Read tab.3. Click .Note: Once disconnected, the software neither monitors norcontrols the 7900HT instrument.to reconnect to an open plate document:1. Select File > Close to close the plate document.2. Click (or select File > Open).3. In the Look In field, navigate to and select the platedocument of interest.4. Click ,Upon opening the plate document, the softwarere-establishes the 7900HT instrument connection.to reconnect to an new plate document, click(or select File > New).Upon creation of the plate document, the softwarere-establishes the connection with the 7900HT instrument.4-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Section 4.3 Automated OperationSection 4.3Automated OperationIn This Section Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-39After the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-44Pre-RunChecklistThe following tasks must be complete to run plates on the 7900HT instrument. Seethe associated page number for details on each procedure.Done Check See PageA background run has been performed in the last week 7-16A pure dye run has been performed in the last 6 months 7-20The instrument tray does not contain a plateIMPORTANT! The instrument tray must be empty to begin a run. Ifthe instrument tray contains a plate, you must eject and remove it.4-44The output stack does not contain plates. –OverviewThe Applied Biosystems 7900HT Fast Real-Time PCR System features the capacityfor high-throughput unattended operation through the use of the AutomationController Software. The Automation Controller Software coordinates the functionof the 7900HT instrument, the fixed-position bar code reader, and the ZymarkTwister Microplate Handler to run batches of prepared plates with minimal userintervention. The Automation Controller Software can run plates using platedocuments stored locally on the computer attached to the instrument, or across anetwork on an SDS Enterprise Database.Options for Automated OperationAs with the SDS Software, the Automation Controller Software requires the creationof a plate document for each plate it runs. The Automation Controller Software canrun batches of plates using plate documents saved to the hard drive of the computerattached to the instrument, or stored on an SDS Enterprise Database.Follow the appropriate procedure for your system:Operating the Software with an SDS Enterprise Database . . . . . . . . . . . . . . . . . . 4-32Operating the Software without an SDS Enterprise Database . . . . . . . . . . . . . . . 4-34Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-31DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentOperating the Software with an SDS Enterprise DatabaseOverviewWorkflowRunning the Automation Controller Software with the SDS Enterprise Database isthe least complicated option for operating the Applied Biosystems 7900HT FastReal-Time PCR System. Plate documents saved to the SDS Enterprise Database areautomatically queued (set up for operation) for use on the Automation ControllerSoftware. Following plate document creation, plates can be prepared and loadedimmediately onto the Zymark Twister Microplate Handler for use.1. Start the Automation Controller Software and log in to the database (seepage 4-32).2. If desired, set the study associations for the relative quantification and allelicdiscrimination runs (see page 4-33).3. Load prepared optical plates and Low Density Arrays onto the stacks of theZymark Twister Microplate Handler (see page 4-41).4. Run the instrument (see page 4-43).5. Analyze the run data.– Allelic Discrimination Analysis (see page 5-5).– Absolute Quantification Analysis (see page 6-5).– Relative Quantification Analysis (see page 6-15).– Dissociation Curve Analysis (see page 6-37).Logging into theAutomationControllerSoftware1. If running the SDS software, do one of the following:• Select Instrument > Disconnect to discontinue communication betweenthe software and the instrument, or• Select File > Exit to close the SDS software.2. Start the Automation Controller Software(select > Programs > Applied Biosystems > SDS 2.2.1 >Automation Controller 2.2).3. In the Login dialog box, enter your user name and password, then clickto log into the database.4. Specify the Session-Study associations as described in “Associating Session andStudy Data” on page 4-33.5. Prepare and load the plates onto the Plate Handler as explained in “LoadingPlates onto the Plate Handler” on page 4-41.4-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Operating the Software with an SDS Enterprise DatabaseAssociatingSession andStudy DataUser Access RequirementIf using the SDS Enterprise Database, all users can associate sessions with studies.However, only users belonging to the Scientist or Administrator User Group cancreate a new study.About the Study AssociationsThe Automation Controller Software can automatically associate the data collectedfrom plate runs (called ‘sessions’) with a specific study. Attaching session data tostudies is optional and can be done later if necessary using the RQ or SNP ManagerSoftware. See “Database Design and Information Management” on page 1-27 formore information about the use of studies.IMPORTANT! After you have attached a session to a study, it cannot you cannot add itto another study until you have unattached it using the RQ or SNP Manager Software.To specify the Study associations:1. In the Automation Controller Software dialog box, do one of the following:To set the study association for:• Relative Quantification runs – Click in the Save RelativeQuantification to Study field (or select Enterprise > Select RQ Study).• Allelic Discrimination runs – Click in the Save AllelicDiscrimination to Study field (or select Enterprise > Select AD Study).Click to set the studyassociation for relativequantification runsClick to set the studyassociation for allelicdiscrimination runs2. In the Select Study dialog box, either:• Select an existing study, or• Click , and configure the Create New Study dialog box, then click.–Name– Enter a name for the study (up to 128 characters).–Creator– (Not editable) Displays your user account name.–Description– Enter a brief description of the study (up to 255 characters).3. Click .4. Prepare and load the plates onto the Plate Handler as explained in “LoadingPlates onto the Plate Handler” on page 4-41.IMPORTANT! The Save Analysis Results check box in the Database Options dialogbox must be selected for the Automation Controller Software to associate sessiondata (default is ON). When the option is selected, the software automaticallyperforms a primary analysis of each plate document it runs, and saves the results tothe database as an analysis session. To access the Save Analysis Results option byselecting Enterprise > Database Options.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-33DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentOperating the Software without an SDS Enterprise DatabaseOverviewWorkflowThe first step in configuring the Applied Biosystems 7900HT Fast Real-Time PCRSystem for automated operation is to add plate documents to the plate queue. Theplate queue is a list of plate document files that the Automation Controller Softwareuses to identify and run associated plates during automated operation. By addingplate documents to the queue, the plate documents automatically become availablefor use with the Automation Accessory.IMPORTANT! After you have added a plate document to the plate queue, thesoftware locks the file, preventing any changes from being made to it until you run orremove the plate document from the queue.The Applied Biosystems 7900HT Fast Real-Time PCR System has three options foradding plate documents to the plate queue. Review the options listed below, thenchoose the method that best suits your needs.• Add a plate document to the plate queue from the SDS software. (page 4-35)• Using the Template Batch utility, create batches of plate documents from a platedocument template and add them to the plate queue. (page 4-36)• Add or remove individual or multiple plate documents to the plate queue usingthe Automation Controller Software. (page 4-39)1. Add plate documents to the plate queue.– Add a plate document to the plate queue using the SDS Software (seepage 4-35)– Create and add plate documents to the plate queue using the TemplateBatch Utility (see page 4-36)2. Start and configure the Automation Controller Software and add or remove platedocuments from the plate queue as needed. (see page 4-39)3. Load prepared optical plates and Low Density Arrays onto the stacks of theZymark Twister Microplate Handler. (see page 4-41)4. Run the instrument. (see page 4-43)5. Analyze the run data.– Allelic Discrimination Analysis (see page 5-5)– Absolute Quantification Analysis (see page 6-5)– Relative Quantification Analysis (see page 6-15)– Dissociation Curve Analysis (see page 6-37)4-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Operating the Software without an SDS Enterprise DatabaseAdding a Plate Document to the Plate Queue (Using the SDS Software)IMPORTANT! A plate document must contain a bar code before you can add it to theplate queue. See page 3-28 for more information on configuring a plate documentwith bar code information.1. In the SDS software, select the Instrument tab.2. In the Instrument tab, select the Queue tab.3. In the Queue tab, click .Have you saved the plate document?– Yes, then the software automatically saves the plate document.– No, do the following:a. In the Look in field of Save As dialog box, navigate to a directory wherethe software can save the new file.b. Select Files of type > SDS 7900HT Document (*.sds).c. Click the File name field, then do one of the following:– Enter a name for the plate document file.– Enter or scan the bar code number of the plate into the field.d. Click . The software saves the plate document.4. When prompted, click to submit the document to the plate queue.After you have added a plate document to the plate queue, the software locks thefile, preventing any changes from being made to it until the plate document isrun or removed from the queue.Note: To release the plate document from the queue, start the AutomationController Software and remove the plate document from the queue, asexplained on page 4-40.5. Click .6. Select File > Close. The SDS software closes the plate document.7. Repeat the procedures in this chapter to create plates and add plates to the queueas needed.8. When you finish creating plate documents, run the queued plates as explained in“Starting and Configuring the Automation Controller Software for Operation”on page 4-39.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-35DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentCreating Plate Documents Using the Template Batch UtilityAbout theTemplate BatchUtilityGenerating PlateDocuments froma TemplateThe Template Batch utility allows you to quickly create multiple plate documentsfrom a single SDS 7900HT Template Document (*.sdt). The Template Batch utilitycan be a useful timesaving device in situations where samples are run on plates withidentical assay configurations.IMPORTANT! Plate documents created by the Template Batch utility do not containsample or plate information (Bar code or Comment settings). You must apply thisinformation to each plate document individually after the file is run.Note: For more information on the elements of the Template Batch dialog box or toview the procedures for importing or editing Plate IDs, click and see the SequenceDetection Systems Software Online Help.1. In the SDS software, open a plate document template from the:Computer Hard Drivea. Click (or select File > Open).b. Select File of type > SDS 7900HT Template Document (*.sdt).c. In the Look in field, navigate to and select the plate document template.d. Click .e. Go to step 2.SDS Enterprise Databasea. Select File > Open Template from Database.b. In the Template Names field of the Select Template dialog box, navigateto and select the plate document template.c. Click .d. Click .e. Save the plate document template to the computer hard drive as explainedon page 3-25.f. Go to step 2.The SDS software displays the plate document template.2. Send the plate document template to the queue:a. Select the Instrument tab.b. Select the Queue tab.3. Click .4. Configure the Template Batch dialog box with Plate IDs:a. Click .4-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Operating the Software without an SDS Enterprise Databaseb. In the Plate ID field of the New Plates dialog box, and scan the bar code ofthe first plate in the batch using the hand-held bar code scanner.LASER HAZARD. Exposure to direct or reflected laserlight can burn the retina and leave permanent blind spots. Never look into thelaser beam. Remove jewelry and anything else that can reflect the beam intoyour eyes. Protect others from exposure to the beam.IMPORTANT! If using the SDS Enterprise Database, the bar codes entered mustbe unique and cannot be used by an existing plate document.c. Repeat step b for every plate in the batch.d. When finished, click .The plate bar codes appear inside the Plate ID field.5. Select a destination directory to store the new plate documents:a. Click .b. In the Look in field, navigate to and select the directory you want to use toreceive the new files.c. Click .The Template Batch dialog box displays the selected destination directory in thePlate Directory field.Destination directory6. Click .The software creates plate documents for all entries listed in the Plate ID list,saves them to the destination directory, adds them to the plate queue, anddisplays a message indicating the number of plate documents it created and sentto the plate queue.7. Click to close the message box.8. Click to close the Template Batch dialog box.9. Select File > Close to close the plate document template.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-37DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the Instrument10. Repeat the procedures in this Chapter 3, “Preparing a Run,” and Chapter 4,“Operating the Instrument,” to create and add additional plates to the queue asneeded.Note: After you have added a plate document to the plate queue, the softwarelocks the file preventing any changes from being made to it until the platedocument has been run or removed from the queue.11. When finished creating and adding plate documents to the queue, run the queueas explained in “Starting and Configuring the Automation Controller Softwarefor Operation” on page 4-39.4-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Operating the Software without an SDS Enterprise DatabaseStarting and Configuring the Automation Controller Software for OperationStarting andConfiguring theSoftwareThe Applied Biosystems 7900HT Fast Real-Time PCR System employs theAutomation Controller Software for automated operation of the 7900HT instrument.The software coordinates the action of the 7900HT instrument, the bar code reader,and the Plate Handler while acquiring and saving raw data during each run.1. If running the SDS software, do one of the following:• Select Instrument > Disconnect to discontinue communication betweenthe software and the instrument, or• Select File > Exit to close the SDS software.2. Start the Automation Controller Software(select > Programs > Applied Biosystems > SDS 2.2.1 >Automation Controller 2.2).3. If using the SDS Enterprise Database, enter your user name and password inthe appropriate fields of the Automation Controller Log In dialog box, thenclick .4. If using the SDS Enterprise Database, specify the Session-Study associations asdescribed in “Associating Session and Study Data” on page 4-33.5. If running the Automation Controller Software using the plate queue, verify thatPlate Queue field contains plate documents for all plates you intend to run.• To add a plate document to the plate queue, see “Adding Plates to thePlate Queue” on page 4-40.• To remove a plate document from the plate queue, see “Removing PlateDocuments from the Plate Queue” on page 4-40.Plate Queue(must contain platedocuments for allplates to be run)6. Prepare and load the plates onto the Plate Handler as explained on page 4-41.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-39DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentAdding Plates tothe Plate QueueRemoving PlateDocuments fromthe Plate Queue1. In the Automation Controller Software, select File > Add Plates.2. In the Look in field of the Open dialog box, navigate to the directory containingthe file or files of interest.3. While pressing and holding the Ctrl key, select the plate document file(s) to addto the plate queue.The software highlights selected files.IMPORTANT! A plate document must contain a bar code before you can add itto the plate queue. See page 3-28 for more information on configuring a platedocument with bar code information.4. Click .The Automation Controller Software adds the plate document(s) to the PlateQueue.Note: After you have added a plate document to the plate queue, the softwarelocks the file preventing any changes from being made to it until the platedocument has been run or removed from the queue.To remove plate documents from the plate queue:To remove…Then…specific platedocuments from theplate queueall plate documentsfrom the plate queue1. While pressing and holding the Ctrl key, select the platedocument files to remove.The software highlights selected files.2. Select File > Clear Selected Plate(s).3. Click .The software removes the selected plate documents from theplate queue.1. In the Automation Controller Software, select File >Clear All Plates.2. Click .The software removes all plate documents from the plate queue.4-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Running Plates Using the Automation Controller SoftwareRunning Plates Using the Automation Controller SoftwareUser AccessRequirementThere is no access requirement. All users can run plates that have been saved to theSDS Enterprise Database.Loading Plates onto the Plate HandlerGuidelinesObserve the following guidelines when loading plates onto the Plate Handler:• Before loading plates onto the Plate Handler, make sure that for each plate:– the associated plate document has been added to the plate queue or iscontained in the SDS Enterprise Database– the plate has been sealed using an optical adhesive cover.• Load the plates into the Plate Handler stacks in any order. The software readsthe bar code of each plate before it is run and matches the plate document andmethod with the plate. The Automation Controller Software can run batches ofup to 84 plates in a single session (21 plates/stack).• Orient the plates inside the stacks so that well A1 ( ) of each plate correspondsto the locations shown in Figure 4-11.• Do not place plates in the output stack (X). The Plate Handler arm uses theempty stack to store plates after it runs them.• If loading Low Density Array(s) onto the Plate Handler:– Load no more than 20 cards per stack.– Pack stacks of cards tightly. (The Plate Handler may fail to detect amisaligned card correctly when loading it.)X1423Zymark Twister MicroplateHandler (top view)Bar codeWell A1Figure 4-11Plate Positions of the Zymark Twister Microplate HandlerApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-41DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentLoading Plates1. Following the guidelines on page 4-41, load the sealed plates or cards into thePlate Handler stacks.2. In the Automation Controller Software, select the check boxes for the platestacks containing plates.IMPORTANT! If you are not using stack 1 or the Restack option explainedbelow, remove all plates from stack 1 before starting the queue. Under thesesettings, the Plate Handler will attempt to stack the run plates from stack 2 in thestack 1 position. If stack 1 contains plates, these settings will cause the PlateHandler to stop the run.Plate stack checkboxes3. If you want to retain the location of the plates in the stacks on the Plate Handler,select the Restack when finished check box.Restack when finished – Instructs the arm to replace a stack of used plates totheir original stack and in their original order after the stack has been run. If theoption is not selected, the arm will place each group of used plates inside thenext vacant stack in clockwise order beginning with the Output stack.Note: Re-stacking plates adds significant operating time when running multipleplates. Use the Restack when finished function only when necessary.4. Begin the plate queue as explained on page 4-43.4-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Running Plates Using the Automation Controller SoftwareRunning the InstrumentStarting thePlate QueueAfter you have loaded all plate documents into the Plate Queue or saved them theSDS Enterprise Database, and you have configured the Instrument Control options,you can start the plate queue.To begin the plate queue, click from the Plate Queue tab.The Automation Controller Software does the following:1. The Zymark Twister Microplate Handler loads the first plate from Stack 1 andplaces it into plate tray for the 7900HT instrument.2. The fixed-position bar code reader scans the bar code for the plate.3. The Automation Controller Software does one of the following:• If linked to the SDS Enterprise Database, the software queries the databasefor the plate document with the bar code of the associated plate. Whenfound, the software automatically downloads the plate document.If the software does not find a plate document with a matching bar code inthe database, the software searches the contents of the plate queue asexplained below.• If not linked to a database, the software searches the plate queue for theplate document with the bar code of the associated plate. If found, theAutomation Controller Software loads the plate document informationfrom the file system for the run.4. The 7900HT instrument loads the instrument tray and runs the plate accordingto the parameters defined by the plate document file.Note: Before starting a real-time run, the instrument may pause (up to15 minutes) to heat the heated cover to the appropriate temperature.If using the SDS Enterprise Database, the Automation Controller Softwareautomatically conducts a primary analysis of each completed run and saves theresults to the database. If you specified a study or studies as explained in“Associating Session and Study Data” on page 4-33, then the softwareautomatically attaches the analyzed session data to the appropriate study.The following options are available during and after the completion of the run:To…See PageMonitor the progress of the run 4-44Stop the runIMPORTANT! If you must stop a run in-progress for any reason,carefully read the instructions on page 4-28 before halting the run.4-44Open the instrument tray (after the run) 4-44Analyze the run data after the run is complete 4-44Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-43DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Chapter 4Operating the InstrumentMonitoringInstrumentProgressThe Automation Controller Software displays the progress of the current run in theThermal Status tab. See page 4-27 for an explanation of the Thermal Status tab.Stopping theInstrumentTo stop the plate queue, clickbox at any time.in the Automation Controller Software dialogIf you clicked ... Then the instrument...while the Plate Handler ishandling a plateafter a plate has been loadedinto the instrument, butbefore the run has started.after the 7900HT instrumenthas started a run.aborts the run and moves the Plate Handler to the homeposition.aborts the current run, ejects the plate, and moves thePlate Handler to the home position.aborts the current run, ejects the plate, and moves thePlate Handler to the home position.IMPORTANT! Stopping a run during thermal cycling canaffect the chemistry of the reactions inside the plate.Before stopping a run, carefully read the guidelines onpage 4-28 to determine the best course of action.Ejecting a PlateTo eject a plate following a halted run (or to open/close the instrument tray),click from the Automation Controller Software window.After the RunAnalyzing theRun DataYou can analyze the run data immediately following the completion of the run.See the appropriate page for your application:Section 5.1 Allelic Discrimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15Section 6.3 Dissociation Curve Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-374-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm

Analyzing End-Point Data 55In This Chapter End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . 5-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-1DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataEnd-Point Runs on the 7900HT InstrumentEnd-Point RunsEnd-point is the term used to describe the category of sequence detection runs inwhich the Applied Biosystems 7900HT Fast Real-Time PCR System is used tomeasure the fluorescence of a biological sample after it has undergone thermalcycling. Unlike real-time runs that can yield quantitative measurements, the focus ofend-point experiments is typically to produce a qualitative result. End-point analysisis commonly used in combination with TaqMan ® reagent-based chemistry to confirmthe presence or absence of specific target nucleic acid sequence in cells, tissues, orfluid samples.Currently, the SDS software supports one type of end-point analysis: AllelicDiscrimination.5-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Notes for Database UsersNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when analyzingAllelic Discrimination data as described in this chapter. This section describes thetypes of actions you may need to perform depending on how your administrator hasconfigured the database. For more information on any of the features describedbelow, see the SDS Enterprise Database for the Applied Biosystems 7900HT FastReal-Time PCR System Administrators Guide.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.When the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 5-1Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.When the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-3DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataDescription of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 5-2Electronic Signature Verification Dialog Box Options5-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Section 5.1 Allelic DiscriminationSection 5.1Allelic DiscriminationIn This Section Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . 5-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-5DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataOverviewAllelicDiscriminationon the 7900HTInstrumentEmploying the5´ NucleaseAssay for AllelicDiscriminationThe Applied Biosystems 7900HT Fast Real-Time PCR System supports allelicdiscrimination using TaqMan ® probes. Allelic discrimination is the process by whichtwo variants of a single nucleic acid sequence are detected in a prepared sample.Allelic discrimination chemistry can be used for single-nucleotide polymorphism(SNP) detection.Allelic discrimination on the 7900HT instrument is made possible through the use ofthe fluorogenic 5´ nuclease assay (see page D-2). During the PCR, the fluorogenicprobes anneal specifically to complementary sequences between the forward andreverse primer sites on the template DNA. Then during extension, AmpliTaq Gold®DNA polymerase cleaves the probes hybridized to the matching allele sequence(s)present in each sample. The cleavage of each matched probe separates the reporter dyefrom the quencher dye, which results in increased fluorescence by the reporter. Afterthermal cycling, the plate is run on the 7900HT instrument, which reads thefluorescence generated during the PCR amplification. By quantifying and comparingthe fluorescent signals using the SDS software, it is possible to determine the alleliccontent of each sample on the plate.Mismatches between a probe and target reduce the efficiency of probe hybridization.Furthermore, AmpliTaq Gold ® DNA polymerase is more likely to displace themismatched probe than to cleave it, releasing the reporter dye. By running the extensionphase of the PCR at the optimal annealing temperature for the probes, the lower meltingtemperatures (T m ) for mismatched probes minimizes their cleavage and consequentlytheir fluorescent contribution. Figure 5-3 illustrates results matches and mismatchesbetween target and probe sequences in TaqMan ® PDARs for AD assays (Livaket al., 1995; Livak et al., 1999).AlleleXProbe-target sequence MatchAlleleYVProbe-target sequence MatchFQQF QProbe-target sequenceMismatchmismatch (higher T m )VQProbe-target sequenceMismatchmismatch (higher T m )VFQLegendVICFAMQuencherAmpliTaqGold DNAPolymeraseGR1556Figure 5-3Target and Probe Sequence InteractionTable 5-1 shows the correlation between fluorescence signals and sequences presentin the sample.Table 5-1Signal and Sequence CorrelationA substantial increase in…Indicates…VIC ® dye fluorescence only homozygosity for Allele X.FAM dye fluorescence only homozygosity for Allele Y.5-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

OverviewTable 5-1Signal and Sequence CorrelationA substantial increase in…both fluorescent signalsIndicates…heterozygosity.AlgorithmicManipulation ofRaw AllelicDiscriminationDataAbout theAutoCallingSystemThe SDS software can analyze raw data immediately upon completion of an allelicdiscrimination run. The term ‘raw data’ refers to the spectral data between 500 nm to660 nm collected by the SDS software during the plate-read. During the analysis, thesoftware employs several mathematical algorithms to generate from the raw data a moredirect measure of the relationship between the spectra changes in the unknown samples.The first mathematical algorithm involves the conversion of the raw data, expressed interms of Fluorescent Signal vs. Wavelength, to pure dye components using the extractedpure dye standards. After the software identifies the dye components, it determines thecontribution of each dye in the raw data using the multicomponent algorithm.After normalization, the software processes all samples associated with markers that areconfigured for AutoCalling using the Applied Biosystems proprietary MaximumLikelihood Algorithm. The algorithm conducts a cluster analysis of the data based on theratio of normalized reporter dye signal (for example, the ratio of FAM to VIC ® dye).The result of the analysis typically yields three major clusters corresponding to the threegenotypic constituents: Allele X homozygous, Allele Y homozygous, and heterozygous.After clustering the data points, the software applies a series of probability tests tofurther refine the clustered data and to identify outliers. For every sample, the softwarecalculates a final “quality value” that represents a measure of the likelihood that thesample belongs to each cluster. The algorithm then applies the quality value for themarker (set in the Analysis Settings dialog box) as a threshold for calling the associatedsample data. Sample-cluster associations that generate quality values greater than thedefined threshold are automatically assigned the call of the associated cluster.Note: Any SNPs which depart substantially from expected Hardy-Weinbergfrequencies should be reviewed.About the 2-Cluster Calling FeatureIf only two clusters are present in a study, the AutoCalling algorithm uses the expectedgenotype frequencies of the clusters to assign their genotypes. If a study contains twolarge clusters and a single sample between the two clusters, the software considers theintermediate sample as an outlier rather than a cluster, and treats the data as a two-clusterstudy.Note: The Maximum Likelihood Algorithm is designed to use No TemplateControls, and autocalling is most effective when NTCs are tasked.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-7DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataCluster VariationsThe SDS software graphs the results of an allelic discrimination run on a scatterplotcontrasting reporter dye fluorescence (Allele X R n versus Allele Y R n ). The softwarerepresents each well of the 384-well plate as a datapoint on the plot. The clustering ofthese datapoints can vary along the horizontal axis (Allele X), vertical axis(Allele Y), or diagonal (Allele X/Allele Y). This variation is due to differences in theextent of PCR amplification, which could result from differences in initial DNAconcentration.The example below shows the variation in clustering due to differences in the extentof PCR amplification.Allele Y homozygotesAllele X/Allele YheterozygotesOutliersAllele X homozygotesNo amplificationFigure 5-4Common Datapoint ClustersGenotypic Segregation of Datapoint ClustersFigure 5-4 illustrates the concept of genotypic segregation of samples within theallele plot. The plot contains four separate, distinct clusters that represent the NoTemplate Controls and the three possible genotypes (allele X homozygous, allele Yhomozygous, and heterozygous). Because of their homogenous genetic compliment,homozygous samples exhibit increased fluorescence along one axis of the plot(depending on the allele they contain). In contrast, heterozygous samples appearwithin the center of the plot because they contain copies of both alleles, and thereforeexhibit increased fluorescence for both reporter dyes.About OutliersSamples that did not cluster tightly may:• Contain rare sequence variations• Contain sequence duplications• Not contain a crucial reagent for amplification (the result of a pipetting error)5-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Before You BeginBefore You BeginUsing the SDSSoftwareOnline HelpExamples inThis SectionFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The illustrations and screenshots that appear within this chapter were created from aplate containing Pre-Developed TaqMan Assays and Reagents for AllelicDiscrimination run to screen 6 human genomic DNA samples (HD 1, 2, 3, 4, 7, 8) for2 targets (CYP 2C9*2 and CYP 2C19*2). Each well of the plate contains 1 µL DNA,TaqMan ® 2✕ Universal PCR Master Mix, forward and reverse primers, and FAM andVIC dye-labeled TaqMan probes.Figure 5-5 illustrates the arrangement of the assays, unknown samples, and notemplate control (NTC) wells on the plate.HD-1 HD-1HD-1HD-1HD-1HD-1HD-1 HD-1HD-1HD-1HD-2 HD-2HD-2HD-2HD-2HD-2HD-2 HD-2HD-2HD-2HD-3 HD-3HD-3HD-3HD-3HD-3HD-3 HD-3HD-3HD-3NTCHD-4 HD-4HD-4HD-4HD-4HD-4HD-4 HD-4HD-4HD-4HD-7 HD-7HD-7HD-7HD-7HD-7HD-7 HD-7HD-7HD-7HD-8 HD-8HD-8HD-8HD-8HD-8HD-8 HD-8HD-9HD-8Not in UseGR2107PDAR CYP 2C9*2PDAR CYP 2C19*2Figure 5-5Configuration of the Samples and Detectors on the Example PlateNote: The probes used in the example experiment were designed using the PrimerExpress ® Primer Design Software and by following the guidelines explained in“Assay Development Guidelines” on page B-2.IMPORTANT! The SDS software does not require that absolute quantification platescontain positive controls.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-9DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataAnalysis ChecklistWhere You Are inthe Procedure 1. Analyze the run data (see page 5-11).2. View the results of the allelic discrimination run (see page 5-13).3. Call allele types for each marker (see page 5-14).4. Scrutinize the allele calls (see page 5-16).5. Choose from the following post-analysis options (see page 5-18):– Reanalyze the run data.– Adjust the display settings for the plate document.– Print elements of the plate document.– Export the plate document results table or plots.5-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Opening the Run DataOpening the Run DataUser AccessRequirementOpening theData from aCompleted RunThere is no access requirement. All users can open allelic discrimination data thathas been saved to the SDS Enterprise Database.Opening a Plate Document File1. Click (or select File > Open).2. In the Look in field, navigate to and select the plate document file.3. Click .The SDS software displays the plate document file.4. Configure the Analysis Settings for each marker as explained below.Opening Un-Analyzed Data from the SDS Enterprise Database1. Click (or select File > Open Document from Database).2. In the Plate Query dialog box, configure the Find Plates Matching TheseCriteria table with parameters that correspond to the document of interest.3. Click .Configurequeries hereClick to beginthe searchDocuments thatmeet the searchcriteria appearhere4. In the Search Results list, scroll to and select the session that you want toanalyze.5. Click .6. Click when finished.7. Configure the Analysis Settings for each marker as explained below.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-11DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataAnalyzing an Allelic Discrimination RunUser AccessRequirementAbout theAnalysisConfiguring theAnalysis SettingsThere is no access requirement. All users can analyze allelic discrimination data.The analysis of SNP (or genotyping) data involves the automatic or manual “calling”of sample data for each marker. The “calls” are data labels assigned to individualsamples to reflect their genomic content. The SDS software can call sample data.The SDS software can call sample data:• Manually, using the toolbar and scatterplot.• Automatically, using the “AutoCalling” system (see “About the AutoCallingSystem” on page 5-7).Before analyzing your data, you must configure the analysis settings for each markerthat you want to analyze using the AutoCalling algorithm. The analysis settings arespecific to each marker and must be set for each one individually.IMPORTANT! You need to configure the analysis settings for a marker only if youwant to use the AutoCalling system to analyze it.To configure the analysis settings:1. Click (or select Analysis > Analysis Settings).2. Do one of the following:To set the analysis settings for:• A single marker, select a marker of interest from the Marker drop-down list.• All markers, select All markers from the Marker drop-down list.3. In the Analysis Settings dialog box:a. Select the Auto caller enabled check box to activate the AutoCallingsystem for the associated marker.b. If you expect the analyzed data to consist of only two clusters, select the2-Cluster Calling check box.c. Enter a percentage value in the Quality field to apply as the quality intervalfor AutoCalling samples.4. Repeat steps 2 through 3 for the remaining markers on the plate document.5. When finished, click .Analyzing theDataDuring the analysis, the software employs several algorithms to generate from theraw data a more direct measure of the relationship between the spectral changes inthe samples.To analyze the data:1. Click (or select Analysis > Analyze).The SDS software analyzes the run data and displays the results in the Results tab.5-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Calling and Scrutinizing Allelic Discrimination DataCalling and Scrutinizing Allelic Discrimination DataAbout the AllelicDiscriminationViewThe SDS software graphs the results of allelic discrimination runs on a scatterplotcontrasting reporter dye fluorescence. After signal normalization andmulticomponent analysis, the software graphs the normalized data from each well asa single data point on the plot. Figure 5-6 illustrates the components of the AllelicDiscrimination plot.Markerdrop-down listCalldrop-down listToolbarScatterplotFigure 5-6Components of the Allelic Discrimination Plot• Marker drop-down list – Determines the marker data that the software displayswithin the plot.• Call drop-down list – When a datapoint is selected, this menu allows you toassign an allele call to the datapoint within the scatterplot.• Toolbar – Contains the following tools for manipulating the plot.IconDescriptionSelects individual data points by clicking or groups of datapoints by clickingand dragging a box across a group of data points.Selects groups of datapoints by encircling them with the tool.Repositions the view within the plot by clicking and dragging the screen.Zooms the plot by clicking the mouse button within the plot or by clickingand dragging a section of the plot to view.Zooms out on the plot by clicking the mouse button within the plot.• Scatterplot – A scatterplot of data points from the run.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-13DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataManually CallingAllele Types1. Select the Results tab.2. In the Allelic Discrimination Plot, zoom out until all crossmarks are visible inthe plot.a. Click (magnifying glass tool).b. Click the plot to zoom out.c. Click (lasso tool).d. Select all of the marks within the plot by clicking and dragging the pointeracross all datapoints in the plot.The software outlines all selected wells within the grid view.e. Examine the tray pane to confirm that all wells are selected. If not all wellsare selected, repeat steps a through d until all wells are visible on the plot.Black border(surrounding selected wells)3. Select the sample cluster exhibiting amplification of the first probe.Allele Xhomozygotes5-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Calling and Scrutinizing Allelic Discrimination Data4. Select Call > Allele X.Select Allele XThe software automatically labels the samples and wells with the Allele X call.Allele Xhomozygotes5. Repeat steps 3 through 4 to apply calls to the rest of the samples within the plot.Call Symbol DefinitionAllele X ● Homozygous for the allele displayed on the associatedaxis of the Allelic Discrimination Plot.Allele Y●Both ● Heterozygous (Alleles X and Y)NTC ■ No Template ControlUndetermined ✕ Unknown (Unlabeled)Note: You can adjust the appearance of the allelic discrimination plot or thedata points it contains using the Display Settings dialog box. See the SequenceDetection Systems Software Online Help for more information.Allele Y homozygotesAllele X/Allele YheterozygotesAllele X homozygotesNo amplification6. If evaluating for multiple markers, do the following:a. In the Marker drop-down list, select a different marker.b. Repeat steps 2 through 5 for the new marker.c. Repeat steps a and b until the alleles for each marker have been called.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-15DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataScrutinizing theAllele Calls1. Verify the calls for the NTC, Allele X, and Allele Y controls.a. In the plate grid, select the wells containing the No Template Controlsamples.The software highlights the datapoints within the allele plot.b. Check that the datapoints cluster in the expected position on the plot.c. If using positive controls, repeat steps a and b for the wells containing theAllele X, and Allele Y controls.Allele Y controlsshould cluster hereAllele X controlsshould cluster hereNo TemplateControls shouldcluster here2. Designate samples that did not cluster tightly as Undetermined.Samples that did not cluster tightly may:• Contain rare sequence variations• Contain sequence duplications• Contain contaminants• Be the result of errors in pipetting or sample preparationUndeterminedsamples5-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Calling and Scrutinizing Allelic Discrimination Data3. Screen for Unknown samples that failed to amplify:a. In the Allelic Discrimination Plot, select the NTC cluster.The SDS software highlights the datapoints within the allele plot and theplate grid.b. In the plate grid, check the wells containing Unknown samples for selectedwells that clustered with the NTCs.Samples that clustered with the No Template Control wells may:• Contain no DNA• Contain PCR inhibitors• Be homozygous for a sequence deletionNTC cluster(selected)No TemplateControl wellsUnknown samplesclustered with the NTCs4. Retest any samples that did not cluster tightly or clustered with NTCs toconfirm the results.5. If evaluating for multiple markers, do the following:a. In the Marker drop-down list, select a different marker.b. Repeat steps 1 through 4 for the new marker.c. Repeat steps a and b until the calls for each marker have been verified.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-17DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point DataAfter the AnalysisUser AccessRequirementPost-AnalysisOptionsChanging theDisplay SettingsPrinting a ReportExporting PlateDocument DataIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save the results of an analysis to the database.The following options are available after the analysis:Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-19Before printing or exporting the analyzed data, you can reconfigure the appearanceof several elements of the plate document including the results table, plate grid, andmost plots (Allelic Discrimination, Raw Data, and Background plots).1. Click (or select View > Display Settings).2. In the Display Settings dialog box, click for further instructions onmodifying the display settings.The SDS software can print a report of the analyzed data containing individual ormultiple elements of the plate document.1. Click (or select File > Print Report).2. In the Print Report dialog box, click the for instructions on setting up,previewing, and printing the report.Exporting Plate Document Data as a Tab-Delimited Text FileThe SDS software can export raw or analyzed data in tab-delimited (*.txt) format forall or a select group of wells on a plate document. The exported files are compatiblewith most spreadsheet applications and programs that can read tab-delimited text.To export run data as a tab-delimited text file, choose one of the following for furtherinstructions:• See “Exporting Plate Document Data” on page A-16.• Click within the table view.Help buttonExporting Plots as GraphicsThe SDS software can export most panes and plots of the plate document as JPEG(Joint Photographic Experts Group) graphic files. The JPEG file format iscompatible with most word processing and spreadsheet applications and can beincorporated directly into HTML documents for viewing by most web browsersoftware.To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or clickwithin the plot of interest for further instructions.5-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

After the AnalysisSaving theResultsThe SDS software saves the results of the analysis differently depending on whetherthe analysis is saved to the SDS Enterprise Database (see below) or to a platedocument file (see page 5-20).Saving the Results to the SDS Enterprise DatabaseIf you use the SDS Enterprise Database, you can save your analysis of the run data tothe database as an analysis session.IMPORTANT! Observe the following when saving sessions to the database:• If you modified the plate document information in any way (by removing wellsfrom use, or by changing the display settings), you must save the plate documentto the database for the changes to persist (select File > Save Plate Document toDatabase).• The SDS Enterprise Database allows two or more users to open and modify datasimultaneously. If another user has opened, analyzed, and saved the same datathat you have just analyzed, the software overwrites the analysis when you saveyours to the database.• Analysis session results are directly linked to the configuration of the platedocument used to create the session. If, after you have saved your analysissession to the database, you modify the linked plate document (by omittingwells, altering sample names, etc.) your analysis session may be 'orphaned' (i.e.,have no relationship to its attached plate document). Consequently, the results ofyour analysis session may change if you reanalyze it because you have changedthe content of the associated plate document containing the raw data.Applied Biosystems recommends the following guidelines for using analysissessions:– Save the plate document as a new document any time you make changes to theplate setup (omit wells, alter sample names, modify detectors or markers, etc.)– Maintain only one saved analysis session per document– Understand that only results sessions saved after the latest changes to a platesetup are “guaranteed” to correspond to the linked plate document.To save the results to the database:1. Select File > Save Results to Database.2. In the Description field of the Save Results to Database dialog box, enter a briefdescription of the plate document (up to 255-characters).Note: The software automatically populates the Session Name field with thename of the associated plate document.3. (Optional) In the Add to Study field, click , then select an existing study,or create a new study by clicking , and doing the following:a. In the Name field of the Configure the Create New Study dialog box, entera name for the study (up to 128 characters).The Creator field is not editable and displays your user name.b. In the Description field, enter a brief description of the study (up to255 characters).c. Click .d. Click .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-19DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Chapter 5Analyzing End-Point Data4. In the Save Results to Database dialog box, click .5. In the confirmation dialog box, click .Saving the SDS 7900HT DocumentAlthough the software save any changes made to the appearance of a plate documentand to the analysis settings, it does not save the calls made during the analysis.1. In the SDS software, select File > Save As.2. In the Look in field of the Save As dialog box, navigate to and select a directoryfor the software to receive the new file.3. In the File name field, do one of the following:• Enter a file name for the plate document file, or• Enter or scan the bar code number for the plate into the field.Note: The SDS software does not require that the file name match the bar codeof the corresponding plate.4. Click .The software saves the plate document to the specified directory.5-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm

Analyzing Real-Time Data 66In This Chapter Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16Essential Experimental Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . 6-23Creating the Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Determining T m Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-1DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when analyzingdata as described in this chapter. This section describes the types of actions you mayneed to perform depending on how your administrator has configured the database.For more information on any of the features described below, see the SDS EnterpriseDatabase for the Applied Biosystems 7900HT Fast Real-Time PCR SystemAdministrators Guide.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.When the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 6-1Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.When the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.6-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Notes for Database UsersDescription of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 6-2Electronic Signature Verification Dialog Box OptionsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-3DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataReal-Time Runs on the 7900HT InstrumentReal-Time RunsQuantitativeRT-PCRAbsoluteQuantificationRelativeQuantificationReal-time is the term used to describe the category of sequence detection runs inwhich the Applied Biosystems 7900HT Fast Real-Time PCR System is used tomeasure the fluorescence of a biological sample during thermal cycling. In contrastto end-point runs, real-time experiments can be used to achieve both qualitative andquantitative measurements. Real-time analysis can be used in combination witheither TaqMan ® or SYBR ® Green 1 double-stranded DNA binding dye reagents for avariety of purposes including quantitative PCR and dissociation curve analysis.Quantitative RT-PCR is a method used to measure small quantities of ribonucleicacid sequences isolated from biological samples. Typical biological samples includecells, tissues, and fluids. During the RT step, reverse transcription of target RNAproduces corresponding complementary DNA (cDNA) sequences. During thesubsequent PCR, the initial concentration of target cDNA is quantified by amplifyingit to a detectable level. The two types of quantitative RT-PCR supported by the7900HT instrument, absolute and relative, are described in detail below.The objective of an absolute quantification experiment is to accurately determine theabsolute quantity of a single nucleic acid target sequence within an unknown sample.The results of an absolute quantification experiment are reported in the same units ofmeasure as the standard used to make them.The objective of relative quantification is to determine the quantity of a singlenucleic acid target sequence within an unknown sample relative to the same sequencewithin a calibrator sample. Relative quantification of target gene expression iscalculated from Applied Biosystems 7900HT Fast Real-Time PCR System datausing one of the following methods:About the Standard Curve MethodThe Standard Curve Method constructs a standard curve similar to that used inabsolute quantification experiments. However, because the goal of relativequantification is to establish a fractional relationship, data produced by relativequantification standards is used differently. For all experimental samples, targetquantity is determined from the standard curve and divided by the target quantity of acalibrator sample. Because the unit from the standard curve drops out, only therelative dilutions of the standard are necessary to determine relative quantities.About the Comparative C T MethodThe SDS software calculates relative levels of gene expression using theComparative C T Method of data analysis. The Comparative C T Method is similar tothe Standard Curve Method except that it uses arithmetic formulas to achieve thesame results for relative quantification. Instead of a standard curve, the ComparativeC T Method calculates relative gene expression using the equation:Relative Quantity = 2 –∆∆C T6-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Section 6.1 Absolute QuantificationSection 6.1Absolute QuantificationIn This SectionThis section contains the following information:Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-5DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataOverviewAbout AbsoluteQuantificationEmploying the5´ NucleaseAssayThe Applied Biosystems 7900HT Fast Real-Time PCR System supports real-timeabsolute quantification of nucleic acids using a standard curve method. The objectiveof absolute quantification is to accurately determine the absolute quantity of a singlenucleic acid target sequence within an unknown sample. The results of an absolutequantification experiment are reported in the same unit of measure as the standardused to make them.Absolute quantification on the 7900HT instrument is accomplished through the useof the polymerase chain reaction and the fluorogenic 5´ nuclease assay (seepage D-2). During setup, standards diluted over several orders of magnitude andunknown samples are loaded onto an optical plate containing master mix andTaqMan reagents targeting a specific nucleic acid sequence. The plate is then loadedinto a 7900HT instrument which has been configured to perform a real-time run.During the thermal cycling, the instrument records the emission resulting from thecleavage of TaqMan ® probes in the presence of the target sequence. After the run, theSDS software processes the raw fluorescence data to produce threshold cycle (C T )values for each sample (see page D-6). The software then computes a standard curvefrom the C T values of the diluted standards and extrapolates absolute quantities forthe unknown samples based on their C T values (see below).Note: See Appendix D, “Theory of Operation,” for more information on thefluorogenic 5´ nuclease assay, real-time data collection, or the mathematicaltransformations of sequence detection data.Figure 6-3 illustrates a standard curve generated from a standard TaqMan ® RNase PInstrument Verification Plate. The arrangement of the samples and standards on theplate are shown in “Examples in This Chapter” on page 6-8.Standard Curve Plot2826C T24Unknowns22201.0 E+2 1.0 E+3 1.0 E+4 1.0 E+5Quantity(LogN initial concentration)Figure 6-3 Standard Curve Generated from a TaqMan RNase P InstrumentVerification Plate6-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

OverviewAlgorithmicManipulation ofRaw DataThe SDS software can analyze raw data immediately after an absolute quantificationrun is complete. The term raw data refers to the spectral data between 500 nm to660 nm collected by the software during the real-time run. During the analysis, thesoftware automatically applies several mathematical transformations to the raw datato generate a more direct measure of the relationship between the spectral changes inthe unknown samples.MulticomponentingThe first mathematical transformation involves the conversion of the raw data,expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye componentsusing the extracted pure dye standards. At the same time, the software determines thecontribution of each dye in the raw data using the multicomponent algorithm.Setting the Threshold and Calling C T sAfter normalization, the baseline and threshold values must be set for the run(see “Kinetic Analysis/ Quantitative PCR” on page D-4 for more information). Theresults of the experiment can be visualized in the Standard Curve graph of theResults tab. The graph consists of a scatterplot of standard and unknown samplesgraphed on a linear-scale plot of Threshold Cycle (C T ) versus quantity value.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-7DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataBefore You BeginUsing the SDSSoftwareOnline HelpExamples in ThisChapterFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The illustrations and screenshots that appear within this chapter were created for aTaqMan ® RNase P 384-Well Instrument Verification Plate (PN 4323306), anexperiment run during the installation of the 7900HT instrument to verify itsperformance. The sealed plate is preloaded with the reagents necessary for thedetection and quantification of genomic copies of the human RNase P gene (asingle-copy gene encoding the RNA moiety of the RNase P enzyme). Each wellcontains pre-loaded reaction mix (TaqMan ® 2✕ Universal PCR Master Mix, RNaseP primers, and FAM dye-labeled probe) and template.Figure 6-4 illustrates the arrangement of standards and samples on the RNase PInstrument Verification Plate. As shown below, the RNase P Instrument VerificationPlate consists of 5 columns of template standards (1250, 2500, 5000, 10,000, and20,000 copies) and two replicate populations (5000 and 10,000 copies).Population 15000NTCSTD 1250STD 2500STD 5000STD 10000STD 20000Population 210000GR2107Figure 6-4Sample Configuration of the Example Plate6-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Analysis ChecklistAnalysis ChecklistWorkflowOverview 1. Open the plate document (see page 6-10).2. Configure the analysis settings for the run (see page 6-11).3. Analyze the run data (see page 6-12).4. For each detector that you configured to determine the baseline and thresholdvalues (see page 6-46):– Automatically – Verify that the baseline and threshold values for the detectorwere set correctly by the software.– Manually – Set the baseline and threshold values for the detector5. Visualize outliers and eliminate any outlying amplification from the run data(see page 6-49).6. View the results of the absolute quantification run (see page 6-14).7. Choose from the following post-analysis options (see page 6-52):– Reanalyze the run data.– Adjust the display settings for the results table, plate grid, and platedocument plots.– Print elements of the plate document.– Export the plate document results table or plots.– Save the analysis.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-9DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataOpening the Run DataUser AccessRequirementOpen aPlate DocumentThere is no access requirement. All users can open absolute quantification data thathas been saved to the SDS Enterprise Database.Opening a Plate Document File1. Click (or select File > Open).2. In the Look in field, navigate to and select the plate document file.3. Click .The SDS software displays the plate document file.4. Configure the Analysis Settings for each marker as explained in “Configuringthe Analysis Settings” on page 6-11.Opening a Plate Document from the SDS Enterprise Database5. Click (or select File > Open Document from Database).6. In the Plate Query dialog box, configure the Find Plates Matching TheseCriteria table with parameters that correspond to the document of interest.7. Click .Configurequeries hereClick to beginthe searchDocuments thatmeet the searchcriteria appearhere8. In the Search Results list, scroll to and select the document that you want toanalyze.9. Click .10. When the software has loaded the plate document, click .11. Configure the Analysis Settings for each marker as explained in “Configuringthe Analysis Settings” on page 6-11.6-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Analyzing the DataAnalyzing the DataUser AccessRequirementConfiguring theAnalysis SettingsThere is no access requirement. All users can analyze absolute quantification datathat has been saved to the SDS Enterprise Database.Before analyzing the plate document data, you must configure the analysis settingsfor each detector that you want to analyze. The analysis settings are detector-specificand must be set for each one individually. For each detector, the SDS software cancalculate baseline and threshold values automatically using the AutoC T algorithm, ormanually using values you supply.Note: You can analyze the run without configuring the analysis settings, however thesoftware will analyze the data using a default baseline of cycle 3 to 15 and a defaultthreshold value of 0.2.1. Click (or select Analysis menu > Analysis Settings).2. In the Detector drop-down list, select a detector.3. In the Analysis Settings dialog box, configure the method (automatic or manual)that the software will use to determine the baseline and threshold values for theselected detectors:To determine the baseline and threshold values:Automatically – Select the Automatic C T option button.IMPORTANT! If you choose to use the AutoC T algorithm, verify that thebaseline and threshold were called correctly for each autocalled detectorfollowing the analysis (use the procedure on page 6-46).Note: See “Automatic Threshold and Baseline Determination” on page 6-18 formore information on the AutoC T algorithm.Manually – Do the following:a. Select the Manual Ct option button.b. In the Threshold field, enter a threshold value to apply to the selecteddetector(s) or leave the field empty and set the threshold value manually asexplained on page 6-46.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-11DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time Datac. Do one of the following. Select the:Automatic Baseline option button to have the software calculate a baselinefor the selected detector(s).Manual Baseline option button, and enter Start and End cycle values toapply to the selected detector(s).Manual Baseline option button, and leave the Start and End cycles fieldsempty. (You will need to set the values manually as explained on page 6-46.)4. If necessary, repeat steps 2 through 3 for any additional detectors.IMPORTANT! The threshold and baseline values set in step 3 on page 6-11 aredetector specific, and must be set for each individual detector used on in theplate document.5. Click to accept the analysis settings and exit.6. Analyze the data as explained below.Analyzing theRunAfter you have configured the analysis settings, you can analyze the run data. Duringthe analysis, the software mathematically transforms the raw data to establish acomparative relationship between the spectral changes in the passive reference dyeand those of the reporter dye. Based on that comparison, the software calculates acycle threshold (C T ) for each reaction (standard and unknown). The software thengenerates a standard curve for the run by plotting the standard samples on a graph ofC T versus initial copy number.Note: See Appendix D, “Theory of Operation,” for a detailed description of the SDSsoftware mathematical transformation of real-time run data.1. Click (or select Analysis > Analyze).The SDS software analyzes the run data and displays the results in the Resultstab.2. Choose from the following:• If using the AutoC T algorithm to set the baseline and threshold values forthe detectors on the plate, verify that the software has called the settingscorrectly for each detector as described on page 6-46,• If you choose to set the baseline and threshold values manually, set themfor each detector on the plate as explained on page 6-46.3. If you choose to remove outliers manually, visualize and eliminate outliers fromthe analyzed run data as explained on page 6-46.6-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Viewing ResultsViewing ResultsViewing theStandard CurveThe software displays the standard curve generated from the run data within theResults tab of the plate document. The standard curve plot displays the unknownsamples on a graph of C T (threshold cycle) vs. Quantity (LogN). Figure 6-5illustrates the components of the standard curve plot.Help buttonDetector drop-down listLegend boxHide Unknowns buttonStandard Curve boxStandard curve plotFigure 6-5Components of the Standard Curve Plot• – Starts the Sequence Detection Systems Software Online Help.• Detector menu – Toggles the data displayed within the plot based on detectorname.• Legend box – Displays a symbol key for the datapoints appearing in the plot.• Hide Unknowns button – Toggles the presence of data from unknown samplesin the plot.• Standard Curve box – Contains the following statistical data describing thestandard curve.– Slope – The slope of the standard curve. The slope of the standard curve isuseful for assessing the efficiency of the assay. At 100% efficiency, a reactionshould achieve a slope of −3.33 since every 10-fold difference in quantitytranslates to a difference of 3.33 C T s.– Y-Inter – The Y-axis intercept of the standard curve.– R2 – The R square value for the standard curve that describes the correlationbetween threshold cycles (C T ) and the log of the quantity value for thesamples that comprise the standard curve plot. The calculation yields a valuebetween 1 and 0, where values closer to 1 indicate better correlation betweenC T and the log of the quantity value.Note: The software calculates the R square value by taking the square of thePearson Coefficient of Correlation (also known as the r value) calculated for thedata points that comprise the plot. The software calculates the R2 value only forthe standards that make up the curve.• Standard curve plot – A scatterplot of datapoints from the absolutequantification run.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-13DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataViewing theAnalysis TableDisplays the results of the absolute quantification calculation in the results table ofthe plate document. Figure 6-6 shows an example of the results table containing thedata from an RNase P Instrument Verification Plate.Help buttonFigure 6-6Analysis Table of the Absolute Quantification Plate DocumentThe columns of the table are:• Position – The coordinate position of the well on the plate.• Sample – The sample name applied to the well.• Detector – The name of the detector assigned to the well.• Task –The task (NTC, Standard, or Unknown) assigned to the well.Note: See page 3-16 for information on applying tasks to the plate document.• C T – The threshold cycle generated by the well during the PCR.Note: The software displays ‘Undetermined’ in the C T column when theassociated sample fails to produce a noticeable rise in fluorescent signal duringthe entire PCR (the fluorescent signal produced by the well never crosses thethreshold defined for the associated detector).• Quantity – For wells containing:– Unknown samples, this column reports the extrapolated quantity value of thesample in the same units of measure as the standard.– Standard samples, this column displays the quantity assigned to the well.Note: See page 3-17 for information on applying quantities to standard wells ofthe plate document.The following two columns contain data only if a well is run as part of a replicate group.• Qty Mean – The arithmetic mean for the quantity values of the replicate groupassociated with the well.• Qty stddev – The standard deviation of the quantity values of the replicategroup associated with the well.• Status – Indicates the analysis status of the sample data.After the AnalysisUser AccessRequirementPost-AnalysisOptionsIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save the analyze data of an absolute quantification run.Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-536-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Section 6.2 Relative QuantificationSection 6.2Relative QuantificationIn This Section Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16Essential Experimental Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . 6-23Creating the Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26Viewing Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-15DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataOverviewRelativeQuantification onthe 7900HTInstrumentThe Applied Biosystems 7900HT Fast Real-Time PCR System supports real-timerelative quantification of nucleic acids using the Comparative C T method. TheComparative C T Method uses the following arithmetic formula to achieve results forrelative quantification:where:–∆∆C2 T∆∆C T =The normalized signal level in a sample relative to the normalized signal levelin the corresponding calibrator sample.Note: The derivation of the above equation is explained in ABI PRISM 7700 SequenceDetection System User Bulletin #2: Relative Quantification of Gene Expression(PN 4303859).Note: The normalized signal levels discussed above refer to the signals generated bythe amplification of the target sequence in the unknown and calibrator samples whichis normalized to the signal generated by the amplification of the endogenous control.Note: The SDS software does not support the Standard Curve Method of relativequantification.Employing the5´ NucleaseAssayRelative quantification on the 7900HT instrument is accomplished through the use ofthe polymerase chain reaction and the fluorogenic 5´ nuclease assay. After carefulselection of an endogenous control, probe and primer sets are designed for both thetarget and control sequences. After the assays are verified, unknown samples areloaded onto an optical plate containing master mix and 5′-nuclease assays targeting aspecific nucleic acid sequence. The reaction plate is then run on a 7900HTinstrument that is configured for real-time analysis.During the run, the instrument records the emission resulting from the cleavage ofTaqMan ® probes in the presence of the target sequence. After the run, the SDSsoftware processes the raw fluorescence data to produce threshold cycle (C T ) valuesfor each sample. The SDS software computes relative quantities from the C T valuesof the calibrator sample and the data from the unknown samples within the geneexpression profile.Note: See Appendix D, “Theory of Operation,” for more information on thefluorogenic 5´ nuclease assay, real-time data collection, or the mathematicaltransformations of sequence detection data.6-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

OverviewAlgorithmic Manipulation of Raw DataThe SDS software can analyze raw data immediately after a relative quantificationrun is complete. The term “raw data” refers to the spectral data between 500 nm to660 nm collected by the software during the real-time run. During the analysis, thesoftware automatically applies several mathematical transformations to the raw datato generate a more direct measure of the relationship between the spectral changes inthe unknown samples.MulticomponentingSetting theThreshold andCalling ThresholdCyclesOutlier RemovalGenerating GeneExpressionValuesThe first mathematical transformation involves the conversion of the raw data,expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye componentsusing the extracted pure dye standards. At the same time, the software alsodetermines the contribution of each dye in the raw data using the multicomponentalgorithm.After multicomponenting, the baseline and threshold values must be set for eachdetector in the study. The software allows you to set the baseline and threshold valuesmanually (see page 6-49) or automatically (see page 6-19). The C T values computedusing the baseline and threshold data are the basis for generating gene expressionvalues.For any PCR, experimental error may cause some wells to amplify insufficiently ornot at all. These wells typically produce C T values that differ significantly from theaverage for the associated replicate wells. If included in the relative quantificationcalculations, these outliers can potentially result in erroneous measurements. Toensure precise relative quantification, replicate groups must be carefully scrutinizedfor outlying wells before the analysis. The software provides options for removingoutliers automatically (see page 6-19) or manually (see page 6-49).The final stage in the analysis is the computation of gene expression values from theC T data. The mathematical process for deriving relative quantification values isbriefly described on page D-8. For detailed information on the derivation of the∆∆C T equation, relative quantification data transformations, or other aspects of5´ nuclease chemistry in relation to relative quantification, Applied Biosystemsrecommends the following references:• For information on performing relative quantification using5´ nuclease chemistry on 7900HT instruments, see:– ABI PRISM 7700 Sequence Detection System User Bulletin #2: RelativeQuantification Of Gene Expression (PN 4303859)– RQ Manager Software User Guide(PN 4351670)– The bibliography of this document for a list of technical papers and research.• For information on the derivation of the ∆∆C T Equation, see:– ABI PRISM 7700 Sequence Detection System User Bulletin #2: RelativeQuantification Of Gene Expression (PN 4303859)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-17DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAutomaticThreshold andBaselineDeterminationThe SDS software features a proprietary AutoC T algorithm that can be used toautomatically generate baseline and threshold values for individual detectors. Thealgorithm calculates baseline and threshold parameters for a detector based on theassumption that the data exhibits the “typical” amplification curve shown inFigure 6-7.Plateau phaseLinear phaseGeometric phase∆R nBackgroundCycleBaselineFigure 6-7Typical Amplification CurveExperimental error (such as contamination, pipetting inaccuracy, and so on) canproduce amplification curves that deviate significantly from the typical data shownabove. The data from these irregularities can affect the AutoC T algorithm by causingit to generate incorrect baseline and threshold parameters for the associated detector.For this reason, Applied Biosystems strongly recommends reviewing all baseline andthreshold parameters determined by the AutoC T algorithm following analysis of thestudy data.6-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

OverviewAutomatic Outlier RemovalAbout OutlyingReplicate WellsAbout theAutomatic OutlierRemovalRegardingAmplificationBeyondReproducibleLimitsFor any PCR, experimental error may cause some wells to amplify insufficiently ornot at all. These wells typically produce C T values that differ significantly from theaverage for the associated replicate wells. If included in the calculations, the C Tvalues from these wells indicate an incorrect level of relative gene expression byskewing the average for the replicate group.The SDS software offers algorithmic identification and removal of outlying data forstudies consisting of replicate populations of three or more wells. The statisticalmethod used by the software is based on Grubbs outlier removal (also known as theMaximum Normalized Residual Test), which permits the exclusion of a single outlierin a population consisting of as few as three replicates.The software applies the Grubbs tests at different stages in the ∆∆C T calculation,depending on the type of endogenous control used in the study. For non-multiplexstudies, the software removes outliers from replicate groups immediately aftercalculating threshold cycles (C T ). For multiplex studies, the software applies thealgorithm following the ∆C T calculation. Also, if an apparent outlier is within 0.25C T s of the mean for the associated replicate group, the software does not remove it.Note: The outlier removal feature is optional and can be activated from the AnalysisSettings dialog box (see page 6-29). Outliers can also be identified and eliminatedmanually as explained on page 6-49.In addition to the Grubbs tests, the algorithm used by the SDS software featuresadditional rules for dealing with SDS data. Unless the majority of samples in areplicate population are at maximum cycle, the algorithm ignores max cycle wells(wells with C T s equal to or greater than the Maximum cycle value).The experimental reproducibility of C T values for relative quantification assaysdecreases as the starting copy number approaches extremely low levels (less than100 copies). Therefore, PCR targets with low starting copy number and subsequenthigh C T values may have significantly diminished statistical reproducibility. To avoidproblems with reproducibility for low-expression targets, the limits of reproducibilitymust be determined experimentally for each relative quantification assay (all targetsand the endogenous control).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-19DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataEssential Experimental ComponentsEndogenousControlCalibratorSampleAll relative quantification experiments require data from a second probe and primerset that amplifies an endogenous control sequence. Endogenous control targets aretypically constitutive RNA or DNA sequences that are present at a statisticallyconsistent level in all experimental samples. By using the endogenous control as anactive reference, the data from the amplification of a messenger RNA (mRNA) targetcan be normalized for differences in the amount of total RNA added to each reaction.Examples of common endogenous controls are: GAPDH, 18S rRNA, and β-Actin.The endogenous control can be amplified independently of the target sequences inseparate wells on the reaction plate (as singleplex or non-multiplex reactions), or inthe same well (as multiplex reactions).IMPORTANT! For non-multiplex experiments, the reactions amplifying theendogenous control must be located on the same plate as the target assays.IMPORTANT! To generate a standard deviation for the relative quantity value of atarget, each plate must contain usable data from at least two replicates of the targetand endogenous control.All relative quantification experiments require data from a calibrator sample. Duringanalysis, the SDS software calculates gene expression levels in samples relative tothe level of expression in a calibrator sample. Thus, the calibrator plays an integralpart in the calculation because it is used as the basis for the comparative results.Examples of possible calibrator samples include:• A zero time-point sample in a time-course experiment• An untreated sample (versus treated samples)• A resting sample (versus activated samples)Note: The SDS software combines the data from replicate calibrator wells at the ∆C Tlevel of the relative quantification calculation (whether the replicates are present on asingle or multiple plates).Replicate WellsFor relative quantification studies, Applied Biosystems recommends the use of threeor more replicate reactions per sample to ensure statistical confidence.Replicates allow you to:• Preserve Data – If an amplification fails in one well, replicate wells canpotentially provide data. This point is especially true in the case of endogenouscontrols which, upon failure, may invalidate the results for the entire plate.• Remove Outliers – The use of replicate populations provide the opportunity forthe visualization and removal of outliers.• Ensure Statistical Reproducibility – In general, the use of replicates ensures agreater degree of experimental reproducibility by providing a means to identifyand refute anomalous data caused by experimental error (such as contamination,pipetting errors, and so on).6-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Getting StartedGetting StartedUsing the SDSSoftwareOnline HelpExamples in ThisChapterFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The screenshots that appear within this chapter were created using a series ofTaqMan ® Cytokine Gene Expression Plates (PN 4304671), a research tool forreal-time, in vitro quantitative evaluation of human cytokine gene expression. Theplate detects the expression of 12 cytokine target sequences and an endogenouscontrol in complementary DNA (cDNA) samples. The TaqMan Cytokine GeneExpression Plate I consists of a MicroAmp ® Optical 96-Well Reaction Plate arrangedinto 24 replicate groups, each containing TaqMan probes and primers for the assay ofone human cytokine mRNA and an endogenous control. Figure 6-8 illustrates theassay configurations for the plate.The study used to produce the screenshots for this section consisted of four plates ofa time-course experiment run to evaluate the expression of 24 target cytokine mRNAover a 36-hour period.36 hrIL-2IL-1αIL-1βPlate 1IL-3 IL-4 IL-5IL-6 IL-7 IL-8Plate 2GR210824 hrIL-10 IL-12p35 IL-12p40GR2108IL-13IL-15IL-17IL-18 G-CSF GM-CSFPlate 312 hrM-CSF IFNγ LTβGR21080 hrTGFβ TNFα TNFΒGR2108Plate 4GR2108Figure 6-8Sample Configuration of the Example PlatesNote: For more information on the TaqMan Cytokine Gene Expression Plate I, seethe TaqMan Cytokine Gene Expression Plate I Protocol (PN 4306744).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-21DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAnalysis ChecklistWorkflowOverview 1. Create an SDS 7900HT Multiple Plate Document (see page 6-23).2. Add relative quantification plates for the analysis (see page 6-24).3. Configure the analysis settings for the run (see page 6-26).4. Analyze the run data (see page 6-30).5. For each detector that you configured to determine the baseline and thresholdvalues (see page 6-46):– Automatically – Verify that the baseline and threshold values for the detectorwere set correctly by the software.– Manually – Set the baseline and threshold values for the detector.6. For each detector that you configured for manual outlier removal, visualize outliersand eliminate any outlying amplification from the run data (see page 6-49).7. View the results of the relative quantification run (see page 6-31).8. Choose from the following post-analysis settings (see page 6-35):– Reanalyze the run data.– Adjust the display settings for the results table, plate grid, and platedocument plots.– Print elements of the plate document.– Export the plate document results table or plots.6-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Options for Analyzing Relative Quantification DataOptions for Analyzing Relative Quantification DataApplied Biosystems provides two ways to analyze data collected for relativequantification experiments.SDS MultiplePlate Documents(for Small-ScaleAnalyses)RQ ManagerSoftware(for Medium toLarge-ScaleAnalyses)In the case of relative quantification, the SDS software uses a different kind of platedocument, called an SDS 7900HT Multiple Plate Document (*.sdm), to analyze datafrom small relative quantification studies consisting of less than 10 plates. Unlikefile-based plate documents, multiple plate documents save the settings and results ofan analysis. Regardless of whether you are analyzing data from a single plate, orfrom multiple plates in a series, you must create a multiple plate document toconduct the analysis.Note: The SDS software does allow you to open individual relative quantificationplate documents, however they cannot be used individually to generate geneexpression values.The RQ Manager Software is a stand-alone application used to conduct medium- tolarge-scale analyses of relative quantification (gene expression) data stored on anSDS Enterprise Database. The software can conduct comparative analyses of over97 detectors (96 target genes and 1 endogenous control) across 200 plates, orapproximately 153,600 multiplexed reactions run on an Applied Biosystems 7900HTFast Real-Time PCR System instrument. See the RQ Manager Software User Guide(PN 4351670) for more information.Creating the StudyCreating aMultiple PlateDocumentTo conduct a comparative analysis of a series of relative quantification plates in astudy, you must first create a Relative Quantification Multiple Plate Document.1. In the SDS software, click (or select File > New).2. Configure the New Document dialog box with the following settings:• Assay – Relative Quantification (∆∆C T ) Study• Container – the plate formats used to run the samples in the study.IMPORTANT! Relative quantification studies cannot compare data from runsperformed using different plate formats (for example, samples run in a 96-wellplate cannot be compared to those run in a 384-well plate).3. Click . The software displays a new plate document with the appropriateattributes.4. Add the relative quantification plate documents to the study as explained in“Adding Plate Documents to the Study” on page 6-24.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-23DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAdding PlateDocuments tothe StudyTo conduct a comparative analysis of a series of relative quantification plates in astudy, you must first add the plates to a Relative Quantification Multiple PlateDocument.Plate documents added to a study must:• Be of the same consumable format (for example, you cannot compare samplesrun on 384-well plates to those run on 96-well plates)• Have been run using the same method (identical thermal cycler conditions,sample volume, and data collection options)• Contain sample names for all In Use wells. (If a plate document does notcontain samples, you can apply sample names to it using the SDS software.)IMPORTANT! Relative Quantification Multiple Plate Documents exclude from theanalysis all wells of plates that do not have a sample name applied to them.IMPORTANT! Relative Quantification Multiple Plate Documents cannot comparedata from runs performed using different plate formats (for example, samples run ina 96-well plate cannot be compared to those run in a 384-well plate).To add plates to the Relative Quantification Multiple Plate Document:1. In the multiple plate document, click .2. In the Add dialog box, do one of the following:If you know where your plate documents are located:a. Select the File tab.b. Navigate to the directory containing the relative quantification platedocument files of interest.c. Press and hold the Ctrl key, select the files of interest, thenclick .Added platedocument filesSelected platedocument filesIf you do not know where your plate documents are located, or want to searchfor specific plate documents:a. Select the Search tab.b. In the Search in field, enter a directory address to search or click and selecta location (directory or hard drive) to search.c. If you want the SDS software to search all subdirectories in the chosenlocation, select Include Subdirectories.6-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Creating the Studyd. In the Plate Query dialog box, configure the Find Plates Matching TheseCriteria table with parameters that correspond to the documents of interest.e. Click .f. In the Search Results list, press and hold the Ctrl key, select the files ofinterest, then click .Plate documentsadded to thestudyQuery designed to findplate documents with filenames containing “RQ“Plate documentsfound by the softwareNote: See “Using the Search Tool” on page A-20 for more information onsearching for plate documents.3. Repeat step 2 to add additional files to the study (up to 10 plates).Note: You can remove plates from the study by selecting the file from the Filelist field, and then clicking .4. When you finish adding files to the study, click .5. Configure the analysis settings for the study as explained in “Configuring theAnalysis Settings” on page 6-26.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-25DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAnalyzing the StudyUser AccessRequirementConfiguring theAnalysis SettingsThere is no access requirement. All users can analyze absolute quantification datathat has been saved to the SDS Enterprise Database.Before analyzing the data for your study, you must specify the analysis settings foreach detector that you want to analyze. The settings are detector specific and must beset for each detector individually. The following sections briefly discuss the analysissettings provided by the software in the Analysis Settings dialog box:• About the Analysis Settings (see below)• Specifying the Analysis Settings (see page 6-29)Note: You can analyze the run without configuring the analysis settings. However,the software analyzes the data using the baseline of cycles 3 to 15 and a defaultthreshold value of 0.2.About the Analysis SettingsC T Analysis forSelected DetectorSettingsThe settings of this group box control the method that the SDS software uses tocalculate the C T s of the sample replicate groups associated with the selected detectorin the Detector drop-down list. As shown in Figure 6-9, the Ct Analysis settingsprovide both automatic and manual methods for determining the baseline andthreshold values.Automatic Ctoption buttonManual Ctoption buttonFigure 6-9C T Analysis for Selected Detectors Settings• Automatic Ct – Instructs the software to calculate the optimal baseline rangeand threshold values for the selected detectors using the AutoC T algorithm(see page 6-18).IMPORTANT! If you choose to use the AutoC T algorithm, Applied Biosystemsrecommends that you verify that the baseline and threshold values are setcorrectly for each detector configured for AutoCalling (see page 6-46).6-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Analyzing the Study• Manual Ct – Instructs the software to use the baseline and threshold valuesspecified in the Analysis Settings dialog box to calculate C T s for the selecteddetectors.The software provides two methods for manually setting the baseline andthreshold values for the detector:– If you know the values that you want to use, enter them into the appropriatefields of the Analysis Settings dialog box.– If you want to set the baseline and threshold using the controls of theAmplification Plot tab, leave the fields empty and set the values later asdescribed on page 6-46.Replicate andOutlier Removalfor Study SettingsThe settings of this group box determine how the software identifies and removesoutlier data for all detectors in the study.Automatic OutlierRemoval check boxOmit replicates whose ∆C T

Chapter 6Analyzing Real-Time DataGene ExpressionSettings for StudySettingsThe settings in this group box determine how the software handles the endogenouscontrol data and RQ Min/Max confidence values for your study. These settings arecrucial to the analysis and are set once for the study.Calibrator Sample drop-down listEndogenous Control Detectordrop-down listControl Type option buttonsRQ Min/Max Confidence drop-down listFigure 6-11Gene Expression Settings for Study Settings• Calibrator Sample – Displays the name of the sample that the software uses asthe calibrator for the study.• Endogenous Control Detector – Specifies the name of the detector that thesoftware will use as the endogenous control for the study.• Control Type – The Multiplexed/Non-Multiplexed option buttons specify howthe software will use the data from the wells labeled with the endogenouscontrol data.• RQ Min/Max Confidence – Specifies the confidence value that the softwarewill use to calculate the standard error of the mean expression level (RQMaxand RQMin values) for the samples in the study.• Export Individual ∆C T s – Instructs the software to export the ∆C T values ofindividual samples in the analysis instead of the average ∆C T values for thereplicate group.6-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Analyzing the StudySpecifying the Analysis SettingsSpecifying theSettings for theAnalysis1. Click (or select Analysis > Analysis Settings).2. In the Detector drop-down list, select:• detector name – To set the Analysis Settings for a specific detector.• All Detectors – To set the Analysis Settings for a all detectorssimultaneously.3. In the Analysis Settings dialog box, configure the method (automatic or manual)that the software will use to determine the baseline and threshold values for theselected detectors:To determine the baseline and threshold values:AutomaticallySelect the Automatic Ct option button. (See page 6-18 for information on theAutomatic Ct algorithm.)If you choose to use the Automatic C T algorithm, verify that the baseline andthreshold were called correctly for each detector configured for AutoCalling(following the procedure on page 6-46).ManuallyDo the following:a. Select the Manual Ct option button.b. In the Threshold field, enter a threshold value to apply to the selecteddetector(s) or leave the field empty and set the threshold value manually asexplained on page 6-46.c. Select one of the following:• Automatic Baseline, to have the software calculate a baseline for theselected detector(s).• Manual Baseline, then enter Start and End cycle values to apply to theselected detector(s).• Manual Baseline, and leave the Start and End cycles fields empty. (Youwill need to set the values manually as explained on page 6-46.)4. If necessary, repeat steps 2 through 3 for any additional detectors.IMPORTANT! The threshold and baseline values set in step 3 are detector specific,and must be set for each individual detector used on in the plate document.5. If evaluating groups of two or more replicates per sample, select or deselect theAutomatic Outlier Removal option.6. If desired, select the Omit Samples Whose ∆C T

Chapter 6Analyzing Real-Time DataAnalyzing the Study Data8. In the Endogenous Control Detector drop-down list, select the detectorrepresenting the endogenous control for your study.IMPORTANT! The detector used as the endogenous control must be present inall sessions attached to the study.9. Select the option button (Multiplexed or Non-Multiplexed) to indicate the typeof endogenous control used.Note: The Multiplexed or Non-Multiplexed option buttons are active only if thesessions loaded for analysis contain both multiplex and non-multiplex reactions.10. In the RQ Min\Max Confidence drop-down list, select the confidence valueyou want the software to use when generating error bars for the gene expressionplot.11. If desired, select the Export Individual ∆C T option button to instruct the SDSsoftware to display the ∆C T values for individual samples belonging to replicategroups. If the option is not selected, the software displays the average ∆C T forthe replicate group.12. Click to accept the analysis settings and exit.13. Analyze the data as explained in “Analyzing the Data” on page 6-30.Analyzingthe DataDuring the analysis, the software mathematically transforms the raw data to establisha comparative relationship between the spectra changes in the passive reference dyeand those of the reporter dye. Based on that comparison, the software calculates acycle threshold (C T ) for each reaction (endogenous control and unknown).Note: See Appendix D, “Theory of Operation,” for a detailed description of the SDSsoftware mathematical transformation of real-time run data.1. Click (or select Analysis > Analyze).The SDS software analyzes the run data and displays the results in theResults tab.2. Choose from the following:• If you choose to set the baseline and threshold values manually, set themfor each detector on the plate as explained on page 6-46.• If using the AutoC T algorithm to set the baseline and threshold values forthe detectors on the plate, verify that the software has called the settingscorrectly for each detector by following the procedure for manually settingthe baseline and threshold on page 6-46 without adjusting the settings.3. If you choose to remove outliers manually, visualize and eliminate outliers fromthe analyzed run data as explained on page 6-46.6-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Viewing ResultsViewing ResultsDisplaying theAnalyzed DataAbout the GeneExpression PlotThe Gene Expression plot displays the data of the selected target detector(s) in theDetectors list. The SDS software allows you to limit the detector and plate datadisplayed in the Gene Expression plot through the Plates and Targets tables of thePlate List Panel. The software displays data from the targets and plates in the listswhose check boxes are selected.The SDS software displays the graphic equivalent of the results of the relativequantification (∆∆C T ) calculations in the Gene Expression plot. The plot is in theupper division of the multiple plate document. Figure 6-12 illustrates the importantcomponents of the plot.Error bar (see below)Arrow (see page 6-31)LegendPlotDetectorsFigure 6-12Components of the Gene Expression Plot• X–Axis – Lists all the targets involved in the analysis. Within each target group,the software displays the relative quantity of the target in each sample.• Y–Axis – The results of the relative quantification calculations are grouped bytarget sequence, and the relative quantities are graphed on a logarithmic scale.The quantities are shown relative to the expression level in the calibrator sample,where each increment corresponds to a 10-fold difference in gene expression.About theExpressionLevelsThe SDS software displays the results of the relative quantification calculations on alogarithmic scale where each increment corresponds to a 10-fold difference in geneexpression. The software displays the expression level for each selected detectorrelative to the expression level in the calibrator sample (shown in the Calibratordrop-down list).Note: Because the calibrator is compared to itself, the expression level for the calibratoralways appears as 1 (1E+00).Table 6-1 on page 6-32 summarizes the circumstances in which the SDS softwaredisplays gene expression levels for sample replicate groups. The shaded rows of thetable indicate the combinations of data for which the software cannot calculateaccurate expression levels.Note: The expression minimum ( ↑) and maximum ( ↓ ) symbols are described indetail on the page 6-33.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-31DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataTable 6-1DetectorSummary of Expression LevelsSample ∆C T sThen expressionExample (for a 40-cycle experiment)Calibrator Testlevel is…Calib ∆C T Test ∆C T ResultTargetEndo< a

Viewing ResultsAbout DownArrowsDown ArrowAbout Up ArrowsUp ArrowAbout theError BarsUndeterminedSamples andOutlier RemovalThe software displays a down arrow to indicate that a level of expression representsthe maximum target gene expression level. The software displays the down arrow ona sample bar when:• The calibrator sample for the detector group yields valid target and endogenouscontrol C T s (C T s less than the maximum cycle), and• A test sample yields an undetermined target C T (and a valid endogenouscontrol).At the levels above, the test sample does not contain enough target cDNA for anaccurate comparison (with the calibrator sample). Therefore, the stated expressionlevel is the maximum possible value for the associated test sample.The software displays an up arrow to indicate that a level of expression represents theminimum target gene expression level. The software displays the up arrow on allassociated sample bars when:• The target sample for the detector group yields valid target and endogenouscontrol C T s (C T s less than the maximum cycle), and• A calibrator sample yields an undetermined target C T (and a valid endogenouscontrol).At the levels above, the calibrator sample does not contain enough target cDNA foran accurate comparison with the test sample. Therefore, the stated expression level isthe minimum possible value for the sample.The software displays error bars for each column in the plot if the associatedexpression level was calculated from a group of two or more replicates. The error barsdisplay the calculated maximum (RQMax) and minimum (RQMin) expression levelsthat represent standard error of the mean expression level (RQ value). Collectively, theupper and lower limits define the region of expression within which the true expressionlevel value is likely to fall. The software calculates the error bars based on the (RQMinand RQMax) confidence interval setting in the Analysis Settings dialog box.Note: Since standard deviation can only be calculated from two or more replicates,the SDS software does not display error bars, RQMin and RQMax values, or standarddeviation (if either the target or the endogenous control has only a single sample).Note: Error bars are a representation of the likelihood of a random sample meetingacceptance criteria based on standard of population, degrees of freedom, and RQMin/Max confidence values. The software does not display error bars for thecalibrator sample because it is compared to itself.The software declares sample as undetermined if the amplification curves for theassociated replicates in the group do not cross the threshold for the detector duringthe duration of the run.• If outlier removal is not turned on, then the software ignores any undeterminedwells and displays the C T of the remaining wells as the average C T .• If outlier removal is turned on, then voting occurs. If the majority of the wells inthe sample replicate group are undetermined, then the software displays theaverage C T as undetermined. If half or more of the wells have valid C T values,then the software ignores the undetermined wells, removes outliers from theremaining wells (if applicable), and averages the resulting data.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-33DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataViewing RelativeQuantities in theAnalysis TableThe software displays the results of the relative quantification calculation in theResults table of the plate document. Figure 6-13 shows an example of the resultstable containing the data from a TaqMan Human Cytokine Plate I.sFigure 6-13Analysis Table of the Absolute Quantification Plate DocumentThe columns of the table are:• Position – Displays the plate the sample is from and the position on the plate(well number)• Sample – Displays the name assigned to the well/sample when the associatedplate was run• Detector – Displays the name of the detector assigned to the sample/wellposition• Task – Displays the task applied to the detector assigned to the sample/wellposition• C T – Displays the average calculated threshold cycle for the replicate groupassociated with the test sample.Note: The software displays Undetermined in the C T column when theassociated sample fails to produce a noticeable rise in fluorescent signal duringthe entire PCR (the fluorescent signal produced by the well never crosses thethreshold level defined for the associated detector).• ∆C T – Displays the normalized threshold cycle for the sample• Avg∆C T – Displays the averaged normalized threshold cycle for the samplereplicate group• ∆C T SD – Displays the number of standard deviations from the average ∆C T ofthe replicate group that the sample’s individual ∆C T value lies• ∆∆C T – Displays the calculated ∆∆C T value for the replicate group associatedwith the test sample.• RQ – Displays the calculated relative level of gene expression for the replicategroup associated with the test sample.• RQ min – Displays the minimum calculated relative level of gene expression inthe test samples• RQ max – Displays the maximum calculated relative level of gene expressionin the test samples• Status – When autocalling is enabled and a sample is reject by the algorithm,the status field indicates how the sample failed the analysis.• Rejected – When autocalling is enabled, indicates if the sample data was rejected.6-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

After the AnalysisAfter the AnalysisUser AccessRequirementPost-AnalysisOptionsSaving theAnalyzed Studyas a MultiplePlate DocumentIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save analyzed data from a relative quantification study.The following options are available after the analysis:Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)After an analysis, the SDS software allows you to save the results as an SDS 7900HTMultiple Plate Document (*.sdm).To save the analysis to an SDS 7900HT Multiple Plate Document (*.sdm):1. In the SDS software, select File > Save As.2. In the Look in field of the Save As dialog box, navigate to and select a directoryfor the software to receive the new file.3. In the File name field, enter a file name for the file4. Click . The software saves the plate document to the specified directory.Saving the Study to the SDS Enterprise DatabaseThe SDS Enterprise Database does not store relative quantification studies(SDS 7900HT Multiple Plate Documents).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-35DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time Data6-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Section 6.3 Dissociation Curve AnalysisSection 6.3Dissociation Curve AnalysisIn This Section Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Determining T m Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-37DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataOverviewAboutDissociationCurve AnalysisEmploying theSYBR Green 1DyeMathematicalTransformationsThe Applied Biosystems 7900HT Fast Real-Time PCR System supports dissociationcurve analysis of nucleic acids using SYBR ® Green 1 double-stranded DNA bindingdye chemistry. The objective of dissociation curve analysis is to accurately determinethe melting temperature (T m ) of a single target nucleic acid sequence within anunknown PCR sample. Typical uses of dissociation curves include detection ofnon-specific products and primer concentration optimization.Dissociation curve analysis on the 7900HT instrument is made possible through theuse of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry(see page D-3). Dissociation curves are commonly performed following the PCRstage of a SYBR Green dye experiment to screen for non-specific products. Togenerate the data needed to create a curve, the 7900HT instrument performs aprogrammed temperature ‘ramp’ in which it slowly elevates the temperature of theplate over several minutes. The specific binding characteristic of the SYBR Green 1Dye permits the 7900HT instrument to monitor the hybridization activity of thenucleic acids present in the sample. During the run, the instrument records thedecrease in SYBR Green fluorescence resulting from the dissociation of dsDNA.After the run, the SDS software processes the raw fluorescence data from the SYBRGreen 1 Dye to generate a more meaningful representation of the relationshipbetween spectral change and temperature for the dissociation curve run.Multicomponenting and NormalizationThe first mathematical transformation involves the conversion of the raw data,expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye componentsusing the extracted pure dye standards. At the same time, the software determines thecontribution of each dye in the raw data using the multicomponent algorithm.Afterwards, the software normalizes the data using the component of the passivereference dye as shown below.R n=R---------------------------------------------( SYBR)R ( PassiveReference)Derivation of Dissociation Curve DataThe SDS software then computes the first derivative of the normalized data (R n ) foreach reading taken by the 7900HT instrument during the temperature ramp. Theresulting derivative data (R n´) is the rate of change in fluorescence as a function oftemperature (see below).dRR n′ = --------- ndTThe software plots the negative of the resulting derivative data on graph of -R n´versus temperature (T) that visualizes the change in fluorescence at each temperatureinterval. The T m for the target nucleic acid can be determined from the graph byidentifying the maximum for the rate of change (displayed as a peak) for theappropriate amplification curve.6-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Before You BeginExample ResultsFigure 6-14 illustrates a typical dissociation curve from an experiment run to detectnon-specific amplification in cDNA samples. The figure displays the dualamplification peaks typical of primer-dimer formation. The amplification from thespecific product is displayed with a T m of 80.5 °C, while the primer-dimer producthas a characteristically lower T m of 74.9 °C.-6.0-5.0-4.0PrimerDimerMain ProductT m =80.5°C-R n-3.0-2.0-1.00.060 65 70 75 80 85 90 95Temperature (°C)Figure 6-14Example of Results from a Dissociation Curve AnalysisBefore You BeginUsing the SDSSoftwareOnline HelpExamples in ThisChapterFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The illustrations and screenshots that appear within this chapter were created from aplate run to determine the purity of a β-actin amplification in unknown samples.Each well of the plate contains SYBR Green 1 dye, forward and reverse primers, andgenomic DNA known to contain complimentary binding sites.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-39DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAnalysis ChecklistWorkflowOverview 1. Analyze the run data (see page 6-41).2. View the results of the dissociation curve analysis (see page 6-42).3. Determine melting temperature (T m ) values for the derivative peaks(see page 6-44).4. Choose from the following post-analysis options (see page 6-44):– Reanalyze the run data.– Adjust the display settings for the results table, plate grid, and platedocument plots.– Print elements of the plate document.– Export the plate document results table or plots.6-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Opening the Run DataOpening the Run DataUser AccessRequirementOpen theUn-analyzedPlate DocumentThere is no access requirement. All users can open dissociation curve data that hasbeen saved to the SDS Enterprise Database.Opening a Plate Document File1. Click (or select File > Open).2. In the Look in field, navigate to and select the plate document file.3. Click .The SDS software displays the plate document file.4. Configure the Analysis Settings for each marker as explained below.Opening Un-analyzed Data from the SDS Enterprise Database5. Click (or select File > Open Document from Database).6. In the Plate Query dialog box, configure the Find Plates Matching TheseCriteria table with parameters that correspond to the session of interest.7. Click .8. In the Search Results list, scroll to and select the session that you want toanalyze.9. Click .10. Click when finished.Analyzing the Run DataUser AccessRequirementAnalyzing theRunThere is no access requirement. All users can analyze dissociation curve data that hasbeen saved to the SDS Enterprise Database.The run data from a temperature ramp can be analyzed immediately following thecompletion of the run. For an explanation of how the software manipulates the rawdata, see “Algorithmic Manipulation of Raw Data” on page 6-7.1. Select all wells in the plate grid.The software outlines the selected wells with a black border.2. Choose one of the following:• Select Analysis > Analyze.• Click .The software analyzes the run data and displays the results in the DissociationCurve tab.3. Determine the T m for the dissociation curves displayed within the DissociationCurve tab as explained on page 6-44.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-41DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataDetermining T m Values for the Analyzed RunViewing AnalyzedDissociationCurve DataThe SDS software displays the results of the dissociation curve analysis within theDissociation Curve tab of the plate document. The tab displays the analyzed data in agraph of the negative of the derivative (-R n´) versus temperature (T) that visualizesthe change in fluorescence at each temperature interval during the ramp.Note: The plot displays data from the selected wells of the plate grid. If you do notsee dissociation curve data, select the wells of the plate grid containing the SYBRGreen dye reactions.Figure 6-15 illustrates the components of the Dissociation Plot.Dissociation Curve tabDissociation PlotDissociation curvesT m display and sliderStep drop-down listPlot drop-down listDetector drop-downFigure 6-15Elements of the Dissociation Plot• Dissociation plot – The plot displays data from the selected wells in the plate grid.Note: The properties of the Dissociation Plot are adjustable. For moreinformation on adjusting the appearance of the plot, click the help button ( )and see the Sequence Detection Systems Software Online Help.• Step drop-down list – Chooses the data displayed within the plot based on theramp.If a plate document contains data from more than one temperature ramp, theStep drop-down list allows you to displays the data from each by selecting theposition of the ramp in the thermal profile.6-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Determining T m Values for the Analyzed Run• T m display and slider – The SDS software displays the T m below the green slider(see below).T mThere are two definitions for the T m value:– The chemical definition is the temperature at which 50% of the DNA is in adouble-stranded configuration.– The mathematical definition is the maximum value for the first derivativecurve within a specific peak.• Plot drop-down list – Chooses the data displayed within the plot based on thederivative calculation. The list offers the following selections:– Raw – When selected, this option plots the normalized reporter fluorescencedata (R n ) on a graph of fluorescence vs. temperature (left-most plot inFigure 6-16).– Derivative – When selected, this option plots derivative data (R n´) on a graphof the derivative vs. temperature (right-most plot in Figure 6-16). Thederivative data is the negative of the rate of change in fluorescence as afunction of temperature.Figure 6-16 shows the plots accessible from the Plot drop-down list.Raw PlotDerivative PlotFigure 6-16Plots of the Dissociation Curve Tab• Detector drop-down list – Chooses the data displayed within the plot based ondetector name.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-43DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataDeterminingT m Values1. Move the pointer over the green T m line located on the Y-axis line of the plot.2. Click and drag the T m line to the maximum point of the derivative plot of interest.The SDS software displays the T m for the curve below the T m line.T m display and sliderNote: The apex of the curvature of represents the maximum rate of change innormalized fluorescence.After the AnalysisUser AccessRequirementPost-AnalysisOptionsIf using the SDS Enterprise Database, you must belong to the Scientist orAdministrator User Group to save analyzed data from a dissociation curve experiment.Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-536-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Section 6.4 Procedure ReferenceSection 6.4Procedure ReferenceIn This Section Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-45DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataSetting the Baseline and Threshold ValuesSetting theBaseline andThreshold Valuesfor the RunIf you choose to set the baseline and threshold values manually for any detector in thestudy, you need to perform the following procedure for each detector.1. Select the Results tab.2. If setting the baseline and threshold for a:Absolute Quantification Plate DocumentSelect all wells of the plate by clicking the upper-left corner of the plategrid.ButtonRelative Quantification Multiple Plate Documenta. Select a plate in the Plates field to view, then select all wells of the plate byclicking the upper-left corner of the plate grid.b. Repeat step a to select all wells of every plate in the study.Click here to select allwells of the plate gridSelect a plate here3. In the Detector drop-down list in the Amplification plot, select a detector forwhich you want to set the baseline and threshold values.The software displays the data for the selected wells within the Amplification plot.4. In the Plot drop-down list, select ∆R n vs. Cycle.5. Double-click the Amplification plot, or click .6-46 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Setting the Baseline and Threshold Values6. In the Display Settings dialog box:a. In the Select Pane/View field, select Amplification Plot.b. In the Y Axis group box of the Scale tab, select the Linear option button.c. Click .7. Identify the components of the linear scale Amplification plot and set thebaseline so that the amplification curve growth begins at a cycle number greaterthan the Stop baseline cycle.If you are inspecting an AutoCalled detector, verify that the baseline is calledcorrectly by the software and proceed to step 8. If the baseline is set incorrectly,open the Analysis Settings dialog box, configure the detector for manualcalling, then click . After the software adjusts the data, manually set thebaseline and threshold for the detector as described in this procedure.If the amplification plot looks like...Then...The amplification curve begins after thedefault maximum baseline (cycle 15).Adjustment of the baseline is notrequired.The maximum baseline is set too high.Decrease the Stop baseline value bydragging the right range marker ( ) toan earlier cycle.The maximum baseline is set too low.Increase the Stop baseline value bydragging the right range marker ( ) to alater cycle.8. Double-click the Amplification plot or click .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-47DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time Data9. In the Display Settings dialog box:a. In the Select Pane/View field, select Amplification Plot.b. In the Y Axis group box of the Scale tab, select the Log option button.c. Click .10. Identify the components of the amplification curve and set the threshold so thatit is:• Above the background• Below the plateaued and linear regions of the amplification curve• Within the geometric phase of the amplification curveIf you are inspecting an AutoCalled detector, verify that the threshold is calledcorrectly by the software and proceed to step 11. If the threshold is setincorrectly, open the Analysis Settings dialog box, configure the detector formanual calling, then click . After the software adjusts the data, manuallyset the baseline and threshold for the detector as described in this procedure.R nPlateau phaseLinear phaseGeometric phaseThreshold setting(click and drag)BackgroundCycleBaseline11. Repeat steps 3 through 10 to set the baseline and threshold values for allremaining detectors present on the plate.12. If you choose to eliminated outliers• Automatically – View the results.• Manually – Visualize and eliminate outliers from the analyzed run data asexplained in “Eliminating Outlying Amplification” on page 6-49.6-48 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Eliminating Outlying AmplificationEliminating Outlying AmplificationOverviewVisualizingOutliersFor any PCR, experimental error may cause some wells to amplify insufficiently or notat all. These wells typically produce C T values that differ significantly from the averagefor the associated replicate wells. If included in the absolute or relative quantificationcalculations, these outliers can potentially result in erroneous measurements.To ensure precise relative quantification, replicate groups must be carefullyexamined for outlying wells. The C T vs. Well Position view of the Amplification plotallows you to examine each set of replicate wells for outliers.1. Select the Results tab.2. If analyzing a Relative Quantification study, select a plate in the Plates field anddeselect all wells in the plate grid. Repeat for each plate in the Plates field.When you are finished, the Amplification plot should not display any data.3. If eliminating outliers from a:Absolute Quantification Plate DocumentSelect all wells of the plate by clicking the upper-left corner of the plate grid.Relative Quantification Multiple Plate Documenta. Select a plate in the Plates field to view.b. Select all wells of the plate by clicking the upper-left corner of the plate grid.4. In the Plot drop-down list, select Ct vs. Well Position.5. In the Well versus Threshold Cycle Amplification plot, verify the uniformity ofeach replicate population by comparing the groupings of C T values for the wellsthat make up the set.Are outliers present?YesNoThen…Record the well numbers of the outlying wells, then go tothe next step.Go to the next step.6. In the Detector drop-down list, select another detector, and repeat steps 3through 5. Repeat until each detector has been screened for outliers.7. If analyzing a Relative Quantification Multiple Plate Document, deselect thewells of the current plate, then repeat steps 3 through 6 for every plate in thestudy.8. Are outliers present on the plate?Yes – If outliers are present for any detector, eliminate the outlying wells fromthe analysis by omitting them as explained in “Eliminating Wells from theAnalysis” on page 6-50.No – If no outliers are present, do one of the following:• If analyzing an Absolute Quantification Plate Document, view the results ofthe run displayed within the Standard Curve plot and Results table.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-49DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time Data• If analyzing a Relative Quantification Multiple Plate Document, view theresults of the run displayed within the Gene Expression plot andResults table.Eliminating Wellsfrom the AnalysisThe SDS software allows you to omit wells from the analysis. If you have identifiedoutliers or data you want to exclude from the analysis, follow the procedure below toremove the corresponding wells from use.IMPORTANT! If a well is removed from use before the plate document is run, theSDS software does not collect data for the well during the run. However, if a well isremoved from use after the plate document has been run, the software excludes thewell from the analysis but does not delete the data.1. If omitting wells from a Relative Quantification Multiple Plate Document,select a plate in the Plates field that contains wells you want to omit.2. While pressing and holding the Ctrl key, select the well(s) in the plate grid thatyou want to omit.3. If omitting wells from a Relative Quantification Multiple Plate Document,repeat steps 1 through 2 for each plate in the study.4. Select the Setup tab.5. In the well inspector, click the Omit Well(s) check box to exclude the selectedwells from the analysis.Selected wells(removed from use)Omit Well(s) check box(selected)Note: To reinstate an omitted well, select the well, then click the Omit Well(s)check box.6-50 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Eliminating Outlying Amplification6. Click (or select Analysis > Analyze).7. Repeat the procedures in “Visualizing Outliers” on page 6-49 to verify that alloutliers have been removed from the analysis.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-51DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time DataAfter the AnalysisUser AccessRequirementPost-AnalysisOptionsChanging theDisplay SettingsPrinting a ReportExporting PlateDocument DataIf using the SDS Enterprise Database, all users can perform the tasks explainedbelow except for saving the results. Only users belonging to the Scientist orAdministrator User Group can save analyzed data to the database as a session.Changing the Display Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Printing a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (see below)Saving the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-53Before printing or exporting the analyzed data, you can reconfigure the appearanceof several elements of the plate document including the results table, plate grid, andmost plots.1. Click (or select View > Display Settings).2. In the Display Settings dialog box, click for further instructions onmodifying the display settings.The SDS software can print a report of the analyzed data containing individual ormultiple elements of the plate document.1. Click (or select File > Print Report).2. In the Print Report dialog box, click for instructions on setting up,previewing, and printing the report.Exporting Plate Document Data as a Tab-Delimited Text FileThe SDS software can export raw or analyzed data in tab-delimited (*.txt) format forall or a select group of wells on a plate document. The exported files are compatiblewith most spreadsheet applications and programs that can read tab-delimited text.To export run data as a tab-delimited text file, choose one of the following for furtherinstructions:• See “Exporting Plate Document Data” on page A-16.• Click within the table view.Exporting Plots as GraphicsThe SDS software can export most panes and plots of the plate document as JPEG(Joint Photographic Experts Group) graphic files. The JPEG file format iscompatible with most word processing and spreadsheet applications and can beincorporated directly into HTML documents for viewing by most web browsersoftware.To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or clickin the plot of interest for further instructions.6-52 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

After the AnalysisSaving theResultsThe SDS software saves the results of the analysis differently depending on whetherthe analysis is saved to the SDS Enterprise Database (see below) or to a platedocument file (see page 6-54).Saving the Analysis Session to the SDS Enterprise DatabaseIf using the SDS Enterprise Database, you can save your analysis of the run data tothe database as an analysis session.IMPORTANT! Observe the following when saving sessions to the database:• If you modified the plate document information in any way (by removing wellsfrom use, or by changing the display settings), you must save the plate documentto the database for the changes to persist (select File > Save Plate Document toDatabase).• The SDS Enterprise Database allows two or more users to open and modify datasimultaneously. If another user has opened, analyzed, and saved the same datathat you have just analyzed, the software overwrites their analysis when you saveyours to the database.• Analysis session results are directly linked to the configuration of the platedocument used to create the session. If, after you have saved your analysissession to the database, you modify the linked plate document (by omittingwells, altering sample names, etc.) your analysis session may be 'orphaned' (i.e.,have no relationship to its attached plate document). Consequently, the results ofyour analysis session may change if you reanalyze it because you have changedthe content of the associated plate document containing the raw data.Applied Biosystems recommends the following guidelines for using analysissessions:– Save the plate document as a new document any time you make changes to theplate setup (omit wells, alter sample names, modify detectors or markers, etc.)– Maintain only one saved analysis session per document– Understand that only results sessions saved after the latest changes to a platesetup are “guaranteed” to correspond to the linked plate document.To save the save the analysis session to the database:1. Select File > Save Results to Database.2. In the Description field of the Save Results to Database dialog box, enter a briefdescription of the plate document (up to 255-characters).Note: The software automatically populates the Session Name field with thename of the associated plate document.3. (Optional) In the Add to Study field, click , then select an existing study,or create a new study by clicking , and doing the following:a. In the Name field of the Configure the Create New Study dialog box, entera name for the study (up to 128 characters).The Creator field is not editable and displays your user name.b. In the Description field, enter a brief description of the study (up to255 characters).c. Click .d. Click .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-53DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Chapter 6Analyzing Real-Time Data4. In the Save Results to Database dialog box, click .5. In the confirmation dialog box, click .Saving the SDS 7900HT DocumentAlthough the SDS software saves the display settings and analysis settings to theplate document, it does not save the results of the analysis. You must analyze theplate document each time you want to review the analysis.1. In the SDS software, select File > Save As.2. In the Look in field of the Save As dialog box, navigate to and select a directoryfor the software to receive the new file.3. In the File name field, do one of the following:• Enter a file name for the plate document file.• Enter or scan the bar code number for the plate into the field.Note: The SDS software does not require that the file name match the bar codeof the corresponding plate.4. Click . The software saves the plate document to the specified directory.6-54 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm

Maintaining the Instrument 77In This Chapter Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4Section 7.1 Maintaining the 7900HT Instrument. . . . . . . . . . . . . . . . . . . . . . . . 7-5Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6Changing the Plate Adapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35Automation Accessory Components and Stack Positions. . . . . . . . . . . . . . . . . . . 7-36Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . 7-37Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . 7-53General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54Maintaining the SDS software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-1DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentNotes for Database UsersDatabase Alertsand NotificationsReason(s) ForChange DialogBoxIf you are using an SDS Enterprise Database to store SDS plate documents, sessions,studies, and data, you may be required to perform additional tasks when performingthe procedures described in this chapter. This section describes the types of actionsyou may need to perform depending on how your administrator has configured thedatabase. For more information on any of the features described below, see the SDSEnterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCRSystem Administrators Guide.The Reason(s) for Change dialog box appears only if the SDS Enterprise Database isconfigured for auditing. The auditing system monitors the creation, modification,and deletion of the SDS data contained by the database.When the Reasons for Change dialog box appears, do one of the following:• Select a description for the change from the drop-down list, then click .• Enter a description of the reason for the change in the field, then click .Reasons for changedrop-down listDefault descriptionsCustom description ofthe reason for the changeFigure 7-1Reasons for Change Dialog Box OptionsElectronicSignatureVerificationDialog BoxThe Electronic Signature Verification dialog box appears only if the SDS EnterpriseDatabase is configured to require electronic signatures. The electronic signaturesystem restricts and tracks user access of the SDS data contained by the database.When the Electronic Signature Verification dialog box appears:1. In the User ID and Password fields, enter your user name and password, thenclick .2. After the software validates your user name and password, it displays a messagestating the success or failure of the signature. Click to continue.7-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Notes for Database UsersDescription of the actionthat requires a signatureEnter your user nameEnter your passwordFigure 7-2Electronic Signature Verification Dialog Box OptionsApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-3DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentRecommended Maintenance ScheduleMaintenanceScheduleThe following table includes a list of tasks that should be performed on theApplied Biosystems 7900HT Fast Real-Time PCR System regularly.Table 7-1Maintenance Schedule for the 7900HT InstrumentInterval Task See PageSu M T W Th F SArchive SDS Files 7-54Week (7 Days)Su M T W Th F SPerform a Background Run 7-16Check (and if necessary replace) Gripper FingerPads7-52Month (30 Days)Defragment the Computer Hard Drive 7-54JanuaryJulyPerform a Pure Dye Run *7-20Su M T W Th F SSu M T W Th F SSemi-Annually(6 Months)Check Applied Biosystems Web Site for SoftwareUpdates7-55*Perform a background run before each Pure Dye Run.7-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Section 7.1 Maintaining the 7900HT InstrumentSection 7.1Maintaining the 7900HT InstrumentIn This Section Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6Changing the Plate Adapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-5DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentReplacing the Sample BlockWhen to PerformSample BlockInstallationWorkflowIMPORTANT! Before changing the sample block, perform all required upgrades tothe SDS software and instrument firmware. Failure to update the software can makethe instrument inoperable or result in damage to instrument components.You need to remove the 7900HT instrument sample block when you:• Decontaminate the wells of the sample block (see page 7-14)• Change sample block formatsIMPORTANT! Always run a background plate after installing the sample block.Unless instructed to do otherwise, adhere to the following guidelines whenexchanging sample block modules of different formats.To install a sample block module:1. Perform all required software and firmware upgrades to the Applied Biosystems7900HT Fast Real-Time PCR System (see page 7-55).2. Remove the existing sample block (see page 7-7).3. Install the new sample block (see page 7-10).4. Change the plate adapter (see page 7-12).5. Run a background plate to check the sample block for contamination(see page 7-16).If you are changing block formats or installing a new block, also do the following:IMPORTANT! Perform pure dye and background runs after replacing the sampleblock, even if you are replacing a sample block module with another block of thesame format.6. Run a pure dye plate or microfluidic pure dye card to create the pure spectracalibration values for the new format (see page 7-20).7. Run the appropriate TaqMan ® RNase P Instrument Verification Plate or aTaqMan ® Low Density 7900HT Installation Array (TGF-β card) to confirm theproper operation of the sample block (see page 7-30).If you are using an Automation Accessory, also do the following:8. If changing consumable formats (for example, when replacing a Standard384-Well Block with a Standard 96-Well Block, a Fast 96-Well Block, or a7900HT System TaqMan ® Low Density Array Upgrade), adjust the plate sensorswitch on the Plate Handler arm for the new plates (see page 7-37).9. Align the Zymark ® Twister Microplate Handler fixed-position bar code readerfor the new plate format (see page 7-41).IMPORTANT! You must align the Plate Handler to only the Instrument position(Zymark position 2) as explained on pages 7-41 to 7-44.10. Align the fixed-position bar code reader for the new plate format(see page 7-49).7-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Replacing the Sample BlockMaterialsRequiredHandling theSample Block• Replacement Sample Block (if replacing the sample block)• 5/32 inch hex key (necessary only for certain instruments)• 5/16 inch hex key (some instruments may require a crescent wrench)The interchangeable sample blocks are delicate pieces of equipment containingseveral fragile components that can break if handled improperly. Figure 7-3 showsthe correct locations for handling the interchangeable sample block module.Circuitry and connections tothe instrument (Do Not Touch)Heat sinks(Bottom of module)GR2028Hold sample blockmodule from the sidesFigure 7-3Locations for Handling Sample Block ModulesRemoving theSample Block1. Start the Automation Controller Software, then:a. Select the Thermal Status tab, then confirm the function of the currentmodule. The module is operating normally if the software is receiving atemperature reading.b. Click to rotate the instrument tray to the OUT position.2. Select File > Exit to close the Automation Controller Software.3. Power off and unplug the 7900HT instrument.PHYSICAL HAZARD. The instrument must be unpluggedand powered off at all times during the following procedure. Failure to complycan result in serious physical injury to the user or damage to the instrument.4. Wait 20 to 30 min for the heated cover to cool.PHYSICAL HAZARD. During instrument operation, thetemperature of the sample block can be as high as 100 °C. Before performingthis procedure, wait until the sample block reaches room temperature.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-7DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

7900HT FAST Real-Time PCR SystemChapter 7Maintaining the Instrument5. If the instrument tray is in the OUT position (outside of the instrument), push itinto the instrument to provide an open workspace.6. If using a Zymark Twister Microplate Handler, remove the covers for thefixed-position bar code reader and the underlying platform.GR20097900HTFront view with RobotGR2009Fixed-position bar code readerand underlying platform covers7. Push the instrument tray inside the instrument, then remove the thermal cycleraccess cover to permit access to the sample block.IMPORTANT! The thermal cycler access cover is secured to the instrument bynon-locking pins and may require force to remove it (no tools are required).GR2023bAccess cover8. Using a 5/16-inch hex key, turn the sample block locking bolt counterclockwiseuntil it is very loose but still attached to the sample block locking bar.IMPORTANT! Some instruments may require the use of an adjustable crescentwrench to loosen the sample block locking bolt.GR2024Sample blocklocking barSample blocklocking bolt7-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Replacing the Sample Block9. Loosen the thumbscrew securing the sample block locking bar to the instrumentchassis (may be a 5/32-inch hex bolt on some instruments).GR2025bGR2024bthumbscrew10. Lift the sample block locking bar up and out of the instrument.11. Remove the sample block from the instrument:a. Rotate the release lever at the base of the sample block 90 degrees.b. Being careful not to damage the heat sinks on the bottom of the sampleblock, slide the sample block out of the instrument and place it on a clean,level surface.GR2027GR2025bApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-9DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentReplacing theSample BlockIMPORTANT! Before changing the sample block, perform all required upgrades tothe SDS software and instrument firmware. Failure to upgrade the software canrender the instrument inoperable or result in damage to instrument components.1. Load the sample block into the instrument compartment:a. Being careful not to damage the heat sinks on the bottom of the sampleblock, rest the sample block on the metal runners on either side of theinstrument bay.b. Carefully slide the sample block into the instrument until the front of theblock is flush with the rear of the locking bar.c. After it is seated, firmly press on the sample block to ensure a goodconnection.2. Replace the sample block locking bar.3. Tighten the thumbscrew (from step 9 on page 7-9) to secure the sample blocklocking bar to the instrument chassis (may be a 5/32-inch hex bolt).4. Using the 5/16-inch hex key, turn the sample block locking bolt clockwise untilit is flush with the locking bar.5. Again, press on the right and left sides of the front surface of the sample blockto ensure that it is seated securely.6. Replace the thermal cycler access cover:a. Fit the lip at the bottom of the access cover over the lower edge of the bay.b. Push the cover towards the instrument until it snaps into place.IMPORTANT! You must reinstall the thermal cycler access cover before poweringon the instrument. Failure to do so prevents the instrument from uploading thefirmware from the computer and causes the SDS software to display an error.GR2023bGR2025bGR20277-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

7900HT FAST Real-Time PCR SystemReplacing the Sample Block7. If using a Plate Handler, replace the covers for the fixed-position bar codereader and the underlying platform (removed in step 6 on page 7-8).GR20097900HTFront view with RobotGR2009Fixed-position bar code readerand underlying platform covers8. Plug in and power on the 7900HT instrument.9. Confirm the function of the installed sample block module:a. Start the Automation Controller Software.b. Select the Thermal Status tab.Does the softwaredisplay temperatures?YesNoThen…the installation is successful.The presence of temperature readings confirm that the7900HT instrument successfully established theconnection to the new sample block.the 7900HT instrument is unable to establishcommunication with the new sample block.To troubleshoot the problem:1. Power off and unplug the 7900HT instrument.2. Remove the thermal cycler access cover.3. Press on the right and left sides of the front plate ofthe sample block to ensure that it is seated securely.4. Reinstall the thermal cycler access cover.5. Repeat step 8 until you hear a high-pitched toneconfirming communication between the instrumentand sample block.10. After the sample block is loaded into the instrument do the following:a. Perform a background run (see page 7-16) to verify that the sample block:– Is connected and working properly– Contains no contaminants that will interfere with fluorescent detectionb. If changing sample block formats, perform any remaining tasks outlined inthe “Sample Block Installation Workflow” on page 7-6.IMPORTANT! Perform pure dye and background runs after replacing the sampleblock, even if you are replacing a sample block module with another block of thesame format.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-11DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentChanging the Plate AdapterWhen to PerformMaterialsRequiredChanging thePlate AdapterRemove and replace the 7900HT instrument plate adapter after changing the sampleblock module format (for example, replacing a Standard 384-Well Block with aFast 96-Well Block).Note: The sample block must be used with the corresponding plate adapter of thesame plate format.• 3/32-inch hex key• One of the following:– 384-Well Plate Adapter– 96-Well Plate Adapter– Fast 96-Well Plate Adapter– TaqMan ® Low Density Array Adapter1. If the instrument tray is inside the 7900HT instrument, move the instrument trayto the OUT position:a. Start the SDS software.b. Click (or select File > New).c. In the New Document dialog box, click .d. In the new plate document, select the Instrument tab.e. In the Real-Time tab of the Instrument tab, click .The instrument tray rotates to the OUT position.f. Select File > Exit. The SDS software exits.2. Remove the four screws attaching the plate holder to the plate arm.UnscrewUnscrew3. Remove the plate adapter from the instrument tray.Note: If changing sample block formats (for example, replacing a Standard384-Well Block with a Fast 96-Well Block), store the plate adapter with thesample block module of the same format.7-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Changing the Plate Adapter4. Place the new plate adapter into the instrument tray with the A1 label in therear-left corner (see below).IMPORTANT! Make sure to install the correct version of the plate adapter forthe plate format you intend to use. The plate adapters are labeled for theconsumable format they support.Well A1Label5. Replace and tighten the four screws in the order shown below:IMPORTANT! The order in which the screws are tightened is important toensure proper alignment of the plate to the sample block inside the 7900HTinstrument.3124Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-13DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentDecontaminating the Sample BlockWhen to PerformMaterialsRequiredThe following procedure describes how to decontaminate the wells of a sample blockmodule. The procedure eliminates residual PCR-related products, includingfluorescent labeled TaqMan ® probes. Clean the sample block as often as needed.IMPORTANT! If you plan to use a cleaning or decontamination method other thanthe one in this manual, check with Applied Biosystems first to ensure that themethod will not damage the sample block module or the 7900HT instrument.• 10% Sodium hypochlorite (bleach) solution• Isopropanol, 100 percent pure• 5/32 in hex key• Cotton swabsCHEMICAL HAZARD. Sodium hypochlorite (bleach) is aliquid disinfectant that can be corrosive to the skin and can cause skindepigmentation. Please read the MSDS, and follow the handling instructions. Wearappropriate protective eyewear, clothing, and gloves.CHEMICAL HAZARD. Isopropanol is a flammable liquid andvapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged orrepeated contact may dry skin and cause irritation. It may cause central nervoussystem effects such as drowsiness, dizziness, and headache, etc. Please read theMSDS, and follow the handling instructions. Wear appropriate protective eyewear,clothing, and gloves.Cleaning a LowDensity ArrayBlockThis procedure applies to the sample block on the 7900HT System TaqMan LowDensity Array Upgrade.1. Wipe the surface of the sample block (grey aluminum) with 10% bleach.2. Wipe the sample block three times with distilled water.3. Wipe the sample block with isopropanol, then allow to air dry.WarningCircuitry and connections tothe instrument (Do Not Touch)Well A-1GR20284. Replace the block as explained in “Replacing the Sample Block” on page 7-10.5. Run a background Low Density Array to confirm that the contamination hasbeen removed.7-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Decontaminating the Sample BlockCleaning aSample BlockThis procedure applies to a:• Standard 384-Well Block• Standard 96-Well Block• Fast 96-Well Block1. Remove the sample block from the 7900HT instrument as explained in“Removing the Sample Block” on page 7-7.2. Use the following figure as a guide to locate the suspected contaminated wellson the sample block.WarningCircuitry and connections tothe instrument (Do Not Touch)Well A-1GR20283. Pipette the appropriate volume of 10% bleach solution into each suspectedcontaminated well of the sample block module.• For a Standard 384-Well Block, pipette 40 µL bleach solution to each well.• For a Standard 96-Well Block, pipette 150 µL bleach solution to each well.• For a Fast 96-Well Block, pipette 55 µL bleach solution to each well.4. Allow the sample block to sit for 3 to 5 min.5. Using a pipet, remove the bleach solution from the wells of the sample block.6. Rinse (pipette and remove) each contaminated well with three treatments ofdeionized water at the appropriate volume for the sample block.• For a Standard 384-Well Block, rinse affected wells with 40 µL deionizedwater.• For a Standard 96-Well Block, rinse affected wells with 150 µL deionizedwater.• For a Fast 96-Well Block, rinse affected wells with 55 µL deionized water.Note: Absolute isopropanol can be substituted for water in the third treatment.7. Remove any remaining isopropanol or water from the wells of the sample blockmodule.8. Replace the sample block as explained in “Replacing the Sample Block” onpage 7-10.9. Run a background plate to confirm that the contamination has been removed.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-15DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentPerforming a Background RunWhen to PerformPurpose ofBackground RunsAbout theBackgroundComponentMaterialsRequiredApplied Biosystems recommends running a background plate monthly or as often asnecessary depending on instrument use.A background run measures the ambient fluorescence in a background plate or LowDensity Array containing deionized water. During the run, the 7900HT instrumentperforms a continuous scan of the plate for 2 minutes at 60 °C. Afterwards, the SDSsoftware averages the spectrum recorded during the run and extracts the resultingspectral component to a calibration file. The software uses the calibration file duringsubsequent runs to remove the background signal from the run data.Because the background signal can change with instrument age, Applied Biosystemsrecommends regenerating the Background component calibration every month.Note: Background runs can also be used to detect and troubleshoot sample blockcontamination.Fluorescence collected by the Applied Biosystems 7900HT Fast Real-Time PCRSystem includes a background component, a fluorescent signal that is inherent to thesystem. The background component is a composite signal found in all spectral datathat consists of fluorescence from several sources including: the backgroundelectronic signal, the sample block, the water inside the consumable, and the plasticconsumable itself. Because the background signal can interfere with the precision ofSDS data, the 7900HT instrument has been engineered to minimize the backgroundsignal. Additionally, the SDS software also algorithmically eliminates thebackground signal from each fluorescent sample to maximize the instrument’ssensitivity.One of the following:• Background plate included in a Spectral Calibration Kit and a centrifuge withplate adapter• Components for making a standard 384- or 96-well background plate:– ABIPRISM 394- or 96-Well Optical Reaction Plate– An optical adhesive cover or ABI PRISM Optical Flat Caps– Pipettor, 100-µL (with pipet tips)– Centrifuge, with plate adapter• Components for making a TaqMan Low Density Array background plate:– Microfluidic card spectral calibration reagents– Centrifuge– Microfluidic card sealer7-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Performing a Background RunPreparing the Background Plate or TaqMan Low Density ArrayPreparing aBackground Card1. Remove the background Low Density Array from its packaging and set it asideto warm to room temperature.2. Remove the tube of background solution from the freezer to thaw. When thesolution has thawed, vortex the tube.3. Load the background solution into the background Low Density Array, loading100 µL of solution per fill reservoir.4. Centrifuge the Low Density Array according to the procedures in “Centrifugingthe TaqMan Low Density Arrays” on page 4-16.5. Seal the Low Density Array according to the procedures in “Sealing theTaqMan Low Density Arrays” on page 4-19.6. Continue with “Creating a Plate Document for the Background Run” below.Preparing andRunning aBackground Plate1. Choose from the following:If…Then…using a backgroundplate from a SpectralCalibration Kitcreating abackground plateremove the plate from the freezer and allow it to thaw toroom temperature.1. Pipette deionized water to each well of the plate.– If using a standard 384-well plate, add 20 µL per well.– If using a standard 96-well plate, add 50 µL per well.2. Seal the plate using an optical adhesive cover or opticalflat caps.2. Briefly centrifuge the background plate.3. Continue with “Creating a Plate Document for the Background Run” below.Creating a Plate Document for the Background RunPreparing aBackground PlateDocument1. Start the SDS software.2. Click (or select File > New).3. Configure the New Document dialog box:• Assay – Select Background.• Container – Select the appropriate plate format.• Template – Select Blank Template.4. If the background plate or Low Density Array is labeled with a bar code, clickthe Barcode field, then scan the bar code number using the hand-held bar codereader.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-17DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument5. Click .The software creates a plate document with the attributes for a background run.IMPORTANT! Do not modify the background plate document. The method for aBackground run is coded into the SDS software and consists of a single hold at60 °C for 2 min. Because the plate contains only deionized water, the platedocument does not require sample or detector labels.6. Save the plate document:a. Click (or select File > Save).b. In the File name field of the Save dialog box, enter:Background_For example, the file name for a background plate run on May 31, 2001would be: Background_053101.c. Click .The software saves the plate document and is now configured for theBackground run.7. Continue with “Running the Prepared Background Plate or TaqMan LowDensity Array” below.Running the Prepared Background Plate or TaqMan Low Density ArrayPerforming theBackground Run1. Load the background plate into the 7900HT instrument:a. In the plate document in the SDS software, select the Instrument tab.b. In the lower portion tab, select the Real-Time tab.c. In the Real-Time tab of the Instrument tab, click .d. Place the background plate or Low Density Array into the instrument trayas shown below.Well A1Position the plate so that the bar codefaces towards the front of the instrumentWell A1Position the Low Density Array so that the barcode faces towards the front of the instrumentNote: The A1 position is in the top-left side of the instrument tray.7-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Performing a Background RunAnalyzing the Background Data2. Click .The 7900HT instrument begins the background run.Note: Before starting the run, the instrument may pause (up to 15 min) to heatthe heated cover to the appropriate temperature.3. When the background run is complete and the Run Complete dialog boxappears:a. Click to close the dialog box.b. Click and remove the plate from the instrument tray.c. Extract the background component as explained on page 7-19.Extracting theBackgroundIn this procedure, you will extract the calibration values from the background platedocument. After extraction, the SDS software stores the data as part of the calibrationfile located in the Calibration subdirectory of the SDS directory.1. Select Analysis > Extract Background.The software attempts to extract the background signal and displays the successof the extraction in a dialog box.If the software displays the:• Calibration Update Complete dialog box, then the run is successful.Proceed to step 2.The raw spectra read from the Background plate conform to acceptablelimits.• Error dialog box, then the run is unsuccessful. Troubleshoot anddecontaminate the sample block as explained in “Background Runs” onpage 8-9.The software has stopped the extraction because one or more raw spectraexceed 2500 FSU.2. Click (or select File > Save).The software saves the plate document.3. Select File > Close.The software closes the plate document.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-19DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentPerforming a Pure Dye RunWhen to PerformPurpose of PureDye RunsComponents ofthe Pure DyeSpectraApplied Biosystems recommends performing spectral calibration:• Every 6 months depending on instrument use• After changing sample block formats (see page 7-6)IMPORTANT! Always run a background plate before performing a Pure Dyecalibration.Pure dye data is generated from the results of a pure dye run in which the SDSsoftware collects spectral data from a set of dye standards during a 2-min hold at60 °C. The software stores the spectral information for the pure dye standards in acalibration file located in the SDS directory. After the run, the software extracts eachcomponent dye spectrum from the collected data in the pure spectra run file.IMPORTANT! Because the age and use of instrument components can affect purespectra readings, Applied Biosystems recommends updating the pure spectra datafiles once or twice annually depending on instrument use.The Applied Biosystems 7900HT Fast Real-Time PCR System monitors fluorescentsignals generated by several dyes inside the preloaded pure dye plate. For thestandard 384-well, standard 96-well, or Fast 96-well pure dye plates, the dyes are:FAM , NED , ROX , SYBR ® Green, TAMRA , TET , and VIC ® .For the 7900HT System TaqMan Low Density Array Upgrade, the 7900HTinstrument monitors fluorescent signals generated by three dyes: FAM , VIC ® , andROX . Your installation kit contains these dyes, and you introduce them into threeseparate microfluidic pure dye cards to perform pure dye runs.1 2 3 4 5 6 7DyePeak (nm)1 FAM ~5202 SYBR Green ~5203 TET ~5404 VIC ~550JOE ~5505 NED ~5706 TAMRA ~5807 ROX ~610Figure 7-4Standard Pure Dye Spectra of the 7900HT InstrumentNote: The 7900HT instrument supports the detection of custom pure dyes (dyesother than those provided by Applied Biosystems). To add custom pure dyes to thePure Dye set for your instrument, see “Adding Custom Dyes to the Pure Dye Set” onpage 7-27.7-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Performing a Pure Dye RunAfter a run, the SDS software receives run data in the form of a raw spectra signal foreach reading. To make sense of the raw data, the software must determine thecontribution of each fluorescent dye used in the sample through a process calledmulticomponenting. The software accomplishes the separation by comparing the rawspectra with a set of pure dye standards contained in a calibration file. When a platedocument is saved after analysis, the software stores the pure spectra informationwith the collected fluorescent data for that experiment.MaterialsRequired384- or 96-Well Plate• Appropriate Spectral Calibration Kit:– Sequence Detection Systems 384-Well Spectral Calibration Kit– ABIPRISM 7900HT Sequence Detection Systems 96-Well SpectralCalibration Kit– 7900HT System Fast 96-Well Spectral Calibration KitNote: Both the standard 96-well and Fast 96-Well Spectral Calibration Kitscontain two pure dye plates.• Product Insert• Centrifuge, with plate adapterMicrofluidic Pure Dye Cards• Microfluidic card spectral calibration reagents• Centrifuge• Microfluidic card sealerApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-21DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentPreparing the Pure Dye Plates or CardsPreparing thePure Dye PlatesPreparing theMicrofluidic PureDye CardsIMPORTANT! A background run must be performed before running a pure dye plate.See “Performing a Background Run” on page 7-16 for more information.1. Remove the pure dye plate from the refrigerator or freezer and set it aside towarm to room temperature.2. Remove the pure dye plate from its packaging.3. Briefly centrifuge the pure dye plate.IMPORTANT! A background run must be performed before running the microfluidicpure dye cards. See “Performing a Background Run” on page 7-16 for moreinformation.1. Remove the three empty microfluidic pure dye cards from the refrigerator andset them aside to warm to room temperature.2. Remove the three dye tubes (FAM, VIC, and ROX dyes) from the freezer tothaw. When the dyes have thawed, vortex the tubes.3. Load the FAM dye into one of the empty microfluidic pure dye cards, loading100 µL of dye per fill reservoir.4. Centrifuge the microfluidic pure dye card per the procedures in “Centrifugingthe TaqMan Low Density Arrays” on page 4-16.5. Seal the microfluidic pure dye card per the procedures in “Sealing the TaqManLow Density Arrays” on page 4-19.6. Repeat steps 3 through 5 for the VIC and ROX dyes.Preparing a Plate Document for a Pure Dye Plate or CardPreparing aPure Dye PlateDocument1. Start the SDS software.2. Click (or select File > New).3. Configure the New Document dialog box:Assay – Select Pure Spectra.Container – Select the appropriate format.Template – If performing a:• Standard 384-Well Pure Dye Run: select 384 Well Pure Dyes Plate.sdt.If no plate document templates are available, construct a Pure Dye platedocument using the product insert from the Spectral Calibration Kit andthe procedure on page 7-28.• Standard 96-Well Pure Dye Run: select the plate document templatematching the Pure Dye plate you intend to run.To run Plate 1 (containing FAM, JOE , NED, and ROX dyes), select96 Well Pure Dyes Plate 1.sdt.To run Plate 2 (containing SYBR, TAMRA, TET, and VIC dyes), select96 Well Pure Dyes Plate 2.sdt.7-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Performing a Pure Dye Run• Fast 96-Well Pure Dye Run: select the plate document template matchingthe Pure Dye plate you intend to run.To run Plate 1 (containing FAM, JOE, NED, and ROX dyes), selectFast 96 Well Pure Dyes Plate 1.sdt.To run Plate 2 (containing SYBR, TAMRA, TET, and VIC dyes), selectFast 96 Well Pure Dyes Plate 2.sdt.• Microfluidic Pure Dye Run: leave the field blank.To run the microfluidic pure dye card containing FAM dye,select MFC FAM Pure Dyes.sdt.To run the microfluidic pure dye card containing ROX dye,select MFC ROX Pure Dyes.sdt.To run the microfluidic pure dye card containing VIC dye,select MFC VIC Pure Dyes.sdt.4. In the Barcode field, scan the bar code number using the hand-held bar codereader.5. Click . The software displays a plate document with the attributes for apure dye run.6. Save the Pure Dye plate document:a. Click (or select File > Save).b. Select Files of type > SDS 7900HT Document (*.sds).c. In the File name field, if running a:• Standard 384-Well Plate, enter PureDye_For example, the file name for a plate run on May 31, 2001 would be:PureDye_053101.• Standard 96-Well Plate, enter PureDye_Plate_For example, the file name for a Pure Dye Plate 1 run on May 31, 2001would be: PureDye_Plate1_053101.• Fast 96-Well Plate, enter FastPureDye_Plate_.sdsFor example, for a fast pure dye plate 1 run on May 31, 2001, type:FastPureDye_Plate1_053101.sds• Microfluidic pure dye card, enter _MFCFor example, the file name for a run performed on May 31, 2001 would be:FAM_053101_MFC.d. Click .The software saves the plate document.7. Prepare and run the pure dye plate or card as explained below.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-23DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentRunning the Prepared Pure Dye Plate or CardRunning thePure Dye Plate orCard1. Load the pure dye plate or microfluidic pure dye card into the 7900HTinstrument:a. In the plate document in the SDS software, select the Instrument tab.b. In the Real-Time tab in the Instrument tab, click .The instrument tray rotates to the OUT position.c. Place the pure dye plate or card into the instrument tray as shown below.Well A1Position the plate so that the bar codefaces towards the front of the instrumentWell A1Position the microfluidic pure dye card so that thebar code faces towards the front of the instrumentNote: The A1 position is located in the top-left side of the instrument tray.2. Click .The 7900HT instrument begins the pure dye run. The method for a pure dye runis coded into the software and consists of a single 2-min hold at 60 °C.Note: Before starting the run, the instrument may pause (up to 15 min) to heatthe heated cover to the appropriate temperature.3. When the pure dye run is complete and the Run Complete dialog box appears:a. Click to close the dialog box.b. Click , and remove the pure dye plate or card from theinstrument tray.c. Extract the pure dye calibration information as explained on page 7-25.7-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Performing a Pure Dye RunAnalyzing the Pure Dye RunExtracting PureDye Informationfrom the RunThe purpose of viewing the data in the Pure Dye Wizard is to eliminate irregular puredye peaks from the data set. The wizard presents the spectral data from the pure dyeplate or card in sets of three wells, each containing the same pure dye. Because thewells displayed by the wizard contain the pure dye at an identical concentration, thesignal peaks for the set should be identical. Occasionally, pipetting inaccuracies orcontamination can cause a well signal to shift slightly. While viewing the data, theoutlying peaks must be eliminated.1. Select Analysis > Extract Pure Dye Wizard.2. Follow the instructions as explained by the Extract Pure Dye Wizard to extractthe pure dye spectra.When presented with each screen, do the following:a. Inspect the spectra for shifts in peak location.Example Spectra from a Pure Dye Plate:WavelengthshiftClick here toremove itExample spectra from a Microfluidic Pure Dye Card:WavelengthshiftClick here toremove itApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-25DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrumentb. If the data set contains an outlying peak, eliminate it by clicking the checkbox of the associated well.Note: Dye spectra are generally acceptable if they peak at the same location astheir group but diverge slightly at other wavelengths.c. Click when finished.d. Repeat steps a to c for all remaining wells until prompted with a messagereporting the extraction of the pure dyes.The software extracts the pure spectra and stores the data as a component of thecalibration file.3. Click (or select File > Save).The software saves the plate document.4. Select File > Close.The software closes the plate document.5. If performing spectral calibration of a:• Standard 384-Well Block – The pure dye calibration is complete.• Standard 96-Well Block – Run the second Pure Dye plate by repeating theprocedures for:– “Preparing a Plate Document for a Pure Dye Plate or Card” on page 7-22– “Running the Prepared Pure Dye Plate or Card” on page 7-24– “Analyzing the Pure Dye Run” on page 7-25• 7900HT System TaqMan Low Density Array Upgrade – Run the secondand third microfluidic pure dye cards containing the VIC and ROX dyes byrepeating the procedures for:– “Preparing a Plate Document for a Pure Dye Plate or Card” on page 7-22– “Running the Prepared Pure Dye Plate or Card” on page 7-24– “Analyzing the Pure Dye Run” on page 7-257-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Adding Custom Dyes to the Pure Dye SetAdding Custom Dyes to the Pure Dye SetWhen to PerformMaterialsRequiredCreating aCustom Pure DyePlateThe Applied Biosystems 7900HT Fast Real-Time PCR System can be used to runassays designed with custom dyes (dyes not manufactured by Applied Biosystems).However, before using custom dyes with the 7900HT instrument, you must createand run a custom pure dye plate.• ABI PRISM 384- or 96-Well Optical Reaction Plate or Optical 96-Well FastThermal Cycling Plate• An optical adhesive cover or ABI PRISM Optical Flat Caps• Custom Dye(s)• Pipettor, 100-µL (with pipet tips)• Centrifuge, with plate adaptorNote: The purpose of the custom pure dye plate is identical to that of an ABI PRISMPure Dye Plate. The SDS software uses the custom plate to create a spectral standardfor multicomponenting the custom dye.1. Prepare a microplate with a dilution series of the custom dye.2. Start the SDS software.3. Create an allelic discrimination plate document and run the dilution series plate.Note: It is not necessary to configure detector, sample, and method information forthe dilution series plate document. The purpose of the run is to establish the correctworking concentration for the dye by viewing the intensity of the raw spectraproduced by the wells in the dilution series.4. Select Analysis > Analyze.The software analyzes the raw run data.5. Click (Show Raw Data Plot).6. In the Raw Data Plot, determine the highest concentration of dye that does notproduce a saturated signal, and record it for future use.Note: Saturated signals are characterized by their high peaks that rise beyonddetectable levels (> 65,000 fluorescent units) and appear as plateaus on theRaw Data plot.The concentration of the custom dye that yields the highest possible signal butdoes not saturate is the maximum concentration for use with the 7900HTinstrument.7. Repeat steps 1 through 6 for any additional custom dyes.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-27DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument8. Create a pure dye plate for the custom dye(s) by pipetting each custom dye to atleast three columns of an optical plate at the concentrations determined instep 7.GR2107Custom Dye 1Custom Dye 2Custom Dye 3Custom Dye 4Custom Dye 5Custom Dye 6Custom Dye 7Custom Dye 8IMPORTANT! The optical configuration of the 7900HT instrument requires thateach pure dye occupy at least three columns of the Pure Dye plate to permitadequate data collection.9. Seal the plate using an optical adhesive cover or optical flat caps.10. Create a plate document template for the custom Pure Dye plate as explainedbelow.Constructing aCustom Pure DyePlate DocumentTemplate1. Start the SDS software.2. Add the new dye to the software using the Dye Manager:a. Select Tools > Dye Manager.b. In the Dye Manager dialog box, click .c. In the Add Dye dialog box, enter a name for the custom dye, andclick .The software adds the new dye to the Custom dye list.d. Repeat steps b and c to add any additional custom dyes to theDye Manager.e. Click .The SDS software makes the new dyes available to pure dye plate documents.3. Create a custom pure dye plate document for the run:a. Click (or select File > New).b. Configure the New Document dialog box with the following options:Assay – Select Pure Spectra.Container – Select the appropriate plate format.Template – Select Blank Template.c. Click . The software creates a new plate document.7-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Adding Custom Dyes to the Pure Dye Set4. Apply pure dyes to the custom plate document:a. Select the wells containing the custom dye.b. In the Dyes drop-down list in the Setup tab, select the appropriate dye.The software applies the dye to the selected wells.c. Repeat steps a and b to configure the plate document with any additionalcustom dyes.Dyes drop-down listCustom dye added to selectedwells of the plate document5. Save the custom Pure Dye plate document as a plate document template:a. Click (or select File > Save).b. In the Save dialog box, navigate to Program Files > AppliedBiosystems > SDS 2.2.1 > Templates.The Templates directory appears inside the Look in field. By saving theplate document template to the Templates directory, it becomes availablefrom the Template drop-down list in the New Document dialog box.c. Select Files of type > SDS 7900HT Template Document (*.sdt).d. In the File name field, and enter a name for the plate document template.e. Click . The software saves the plate document as a plate documenttemplate.6. Run the custom Pure Dye plate as explained in “Preparing the Pure Dye Plates”on page 7-22.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-29DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentVerifying Instrument PerformanceWhen to PerformPurpose ofRNase P RunsApplied Biosystems recommends performing an RNase P or TGF-β run:• When changing sample block formats for the first time• As needed to verify the function of the 7900HT instrumentThe TaqMan RNase P Instrument Verification Plates are experiments run to verifythe performance of the 7900HT instrument for use with optical plates of the sameformat (384-Well, 96-Well, or 96-Well Fast). The TaqMan RNase P InstrumentVerification Plates are pre-loaded with the reagents necessary for the detection andquantification of genomic copies of the human RNase P gene (a single-copy geneencoding the RNase moiety of the RNase P enzyme). Each well contains pre-loadedreaction mix (master mix, RNase P primers, and FAM-labeled probe) and template.Table 7-2 illustrates the arrangement of standards and samples on each type ofTaqMan RNase P Instrument Verification Plate. As shown below, the TaqMan RNaseP Instrument Verification Plates consist of five columns of template standards (1250,2500, 5000, 10,000, and 20,000 copies) and two unknown populations (5000 and10,000 copies).Table 7-2 Configurations of the TaqMan RNase P Instrument Verification PlatesTaqMan RNase PInstrument VerificationPlateSample ConfigurationTaqMan ® RNase P 384-WellInstrument VerificationPlate (PN 4323306)Unknown 15000GR2107NTCSTD 1250STD 2500STD 5000STD 10000STD 20000Unknown 2100007-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Verifying Instrument PerformanceTable 7-2 Configurations of the TaqMan RNase P Instrument Verification PlatesTaqMan RNase PInstrument VerificationPlateTaqMan ® RNase PInstrument VerificationPlate(PN 4310982, standard96-well plate)Sample ConfigurationUnknown 15000NTC STD 1250 STD 2500STD 5000 STD 10000 STD 20000Unknown 210000GR2108Purpose ofTGF-β RunsMaterialsRequiredThe TaqMan ® Low Density 7900HT Installation Array (TGF-β card) is anexperiment run to verify the performance of the 7900HT instrument for use withTaqMan Low Density Arrays. The TGF-β card is preloaded with the reagentsnecessary for the detection and quantification of genomic copies of the TGF-β gene.Each well contains preloaded TGF-β primers and FAM MGB dye-labeled probe.RNase P Runs• Appropriate TaqMan RNase P Instrument Verification Plate (see Table 7-2 onpage 7-30)• Centrifuge, with plate adaptorTGF-β Runs• TGF-β card• TaqMan ® 2✕ Universal PCR Master Mix• Water, DNase/RNase-free, deionized• 2-mL microtube, sterile• Centrifuge• Microfluidic card sealerApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-31DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentPreparing theVerification Plateor TGF-β CardPreparing a TaqMan RNase P Instrument Verification Plate1. Remove the TaqMan RNase P Instrument Verification Plate from the freezerand allow it to thaw to room temperature.2. Briefly centrifuge the TaqMan RNase P Instrument Verification Plate.3. Continue with “Preparing the Plate Document” on page 7-33.Preparing a TGF-β Card4. Remove the TGF-β card from the refrigerator and allow it to warm to roomtemperature.5. In a sterile 2-mL tube, prepare the following PCR reaction mixture:ReagentVolume (µL)TaqMan 2✕ Universal PCR Master Mix 500Human cDNA 50RNase/DNase-free water 450Total Volume 10006. Tap the tube gently to mix.7. Pipette 100 µL of the reaction mix into each of the eight fill reservoirs.8. Continue with “Preparing the Plate Document” on page 7-33.7-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Verifying Instrument PerformancePreparing thePlate Document1. Start the SDS software.2. Click (or select File > New).3. Configure the New Document dialog box.Assay – Absolute QuantificationContainer – Select the appropriate plate format.Template – If running a:• TaqMan ® RNase P 384-Well Instrument Verification Plate, select384 Well RNaseP Install Plate.sdt.• TaqMan ® RNase P Instrument Verification Plate (standard 96-well plate),select 96 Well RNaseP Install Plate.sdt.• TaqMan ® RNase P Fast 96-Well Instrument Verification Plate, select96 Well RNaseP Install Plate.sdt.• TGF-β card, select rnase p card template.sdt.Note: If no plate document templates are available, construct a Pure Dye platedocument using the product insert from the Spectral Calibration Kit and theprocedure on page 7-28.4. If desired, enter the bar code information into the plate document:a. Click the Barcode text field.b. Remove the TaqMan RNase P Instrument Verification Plate or TGF-β cardfrom the packaging and scan its bar code using the hand-held bar codereader.5. Click . The software creates a plate document.Note: Do not modify the plate document. The plate document template ispre-programmed with detector and method information for the run.6. Save the plate document:a. Click (or select File > Save).b. In the Barcode field of the Save dialog box do one of the following:– Enter a name or bar code number for the plate, then click .– Using the hand-held bar code reader, scan the bar code number.c. Select Files of type > SDS 7900HT Document (*.sds).d. Click .The software saves the plate document. The software is now configured for theRNase P run.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-33DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentRunning theVerification Plateor TGF-β CardVerifyingInstrumentPerformance1. In the plate document in the SDS software, select the Instrument tab.2. In the lower portion of the Instrument tab, select the Real-Time tab.3. If the instrument tray is inside the 7900HT instrument, click .The instrument tray rotates to the OUT position.4. Place the TaqMan RNase P Instrument Verification Plate or TGF-β card into theinstrument tray.Note: The A1 position is located in the top-left corner of the instrument tray.5. In the Real-Time tab, click . The 7900HT instrument begins the run.Note: Before starting the PCR run, the instrument may pause (up to 15 min) toheat the heated cover to the appropriate temperature.6. When the run is complete:a. Analyze the run data as explained on page 6-12.b. Set the baseline and threshold values for the analyzed data as explained onpage 6-46.c. Verify the performance of the 7900HT instrument as explained below.Analyzing Data from RNase P RunsThe install specification of the Applied Biosystems 7900HT Fast Real-Time PCRSystem demonstrates the ability to distinguish between 5,000 and 10,000 genomeequivalents with a 99.7% confidence level for a subsequent sample run in a singlewell.The following equation verifies the 7900HT instrument install specifications:[( CopyUnk 1)–3( σ CopyUnk1)] > [( CopyUnk 2)–3( σ CopyUnk2)]where:• CopyUnk 1 is the Average Copy Number of Unknown #1 (10,000 replicatepopulation)• σ CopyUnk1 is the Standard Deviation of Unknown #1 (10,000 replicate population)• CopyUnk 2 is the Average Copy Number of Unknown #2 (5000 replicatepopulation)• σ CopyUnk2 is the Standard Deviation of Unknown #2 (5000 replicate population)Note: The values above can be obtained from the experimental report window.Note: Up to six wells from each replicate group in a 96-well TaqMan RNase PInstrument Verification Plate (both standard 96-well and Fast 96-well plates) can beignored to meet specifications.Note: Up to 10 wells from each replicate group in a 384-well TaqMan RNase PInstrument Verification Plate can be ignored to meet specifications.7-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Section 7.2 Maintaining the Plate HandlerSection 7.2Maintaining the Plate HandlerIn This Section Automation Accessory Components and Stack Positions. . . . . . . . . . . . . . . . . . . 7-36Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . 7-37Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-35DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentAutomation Accessory Components and Stack PositionsAutomationAccessoryComponentsRefer to Figure 7-5 for the components discussed in this section.(cross-sectional view of the gripper)GR2014aPlate-sensorswitchGripperAdjustmentknobPlate stackExpansion stacksZymark Twister Microplate HandlerFixed-position bar code readerFigure 7-5Components of the Automation AccessoryPlate StackPositionsThe Zymark Twister Microplate Handler alignment is performed using the Zymark ®Twister Software. The software refers to the positions of the plate stacks differentlythan the Automation Controller Software. Figure 7-6 lists the positions defined bythe Zymark Twister Software and the Automation Controller Software equivalents.107Zymark TwisterSoftwareAutomationControllerSoftware26Position 0Output345Position 1Position 2(unused)Instrument(front of instrument)Bar codeWell A1Position 3(unused)Position 4 Stack 1Figure 7-6Positions of the Automation Controller and Zymark Twister Software7-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Adjusting the Sensitivity of the Plate Sensor SwitchAdjusting the Sensitivity of the Plate Sensor SwitchWhen to PerformMaterialsRequiredAdjusting thePlate SensorSwitchThe plate sensor switch located underneath the arm of the Zymark TwisterMicroplate Handler requires adjustment under the following circumstances:• When changing sample block module formats• If the Plate Handler is having difficulty sensing plates• Optical plate (of the current sample block format)The dimensions of different plate formats can place different requirements on howthe Plate Handler grips plates. To ensure smooth operation of the AutomationAccessory, adjust the plate sensor switch when changing consumable formats.1. Power off the Zymark Twister Microplate Handler.PHYSICAL HAZARD. The Zymark Twister MicroplateHandler must be powered off at all times during the following procedure. Failureto comply can result in physical injury to the user or damage to the PlateHandler.2. Clear the switch position by turning the thumb wheel all the way to the Upextreme (as indicated on the side panel).Thumb wheelGR2014aPlate-sensor switch3. Begin the adjustment of the sensor switch:a. Grasp an optical plate by the sides, making sure not to place pressure in thecenter of the plate to deform it.b. Place the plate between the fingers of the gripper assembly and align it tothe middle of the centering device.c. While holding the plate in position, slowly turn the thumb wheel to lowerthe switch onto the reaction plate until the switch:– Contacts the top of the plate, and– Emits a soft, audible “clicking” noiseIMPORTANT! The sound emitted by the sensor switch is very faint and may bedifficult to hear. To make the adjustment easier, place your ear close to thesensor switch while making the adjustment and listen for the switch to engage.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-37DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument4. Remove the plate and listen for the plate-sensor switch to disengage.Did you hear theswitch disengage?NoYesThen…1. Move the switch Down a few steps by turning the thumbwheel in the direction indicated on the arm.2. Replace the plate inside the gripper and listen for theswitch to engage:– If you do not hear the switch engage, then remove theplate and repeat steps 1 and 2 above.– If you hear the switch engage, remove the plate andcontinue to step a below.1. Move the switch Up by turning the thumb wheel one stepin the direction indicated on the arm.2. Replace the plate and listen for the switch to engage:– If you hear the switch engage, remove the plate andrepeat steps 1 and 2 above.– If you do not hear the switch engage, then you havesuccessfully identified the zero point of theplate-sensor switch.Note: At the zero point, one step of the thumb wheel in theDown direction causes the switch to engage.5. After the zero point is established, carefully turn the thumb wheel in the Downdirection the number of steps appropriate for your plate format as indicatedbelow:ConsumableABI PRISM 96-Well OpticalReaction PlateABI PRISM 384-Well OpticalReaction PlateTaqMan Low Density ArrayTurn the thumb wheel in the Down direction…20 steps15 steps15 stepsNote: If you lose count, begin again from step 4 and identify the zero point forthe switch.6. Test the adjustment as explained on page 7-39.7-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Adjusting the Sensitivity of the Plate Sensor SwitchTesting theAdjustment1. Place the reaction plate in the input stack 1 of the Plate Handler.2. Power on the 7900HT instrument, the Plate Handler, and the computer.3. Select > Programs > Zymark Twister Plate Handler > Twister.The Zymark Twister Software starts.4. Click Manual Control.5. In the Manual Control dialog box, click stack 4.ClickThe Plate Handler arm moves over the input stack.6. Click .If the adjustment was successful, the Plate Handler arm will lower upon theplate until the plate detector switch engages confirming the presence of theplate.If Plate Handler arm emits a grinding sound, adjust the plate sensor switch:a. In the Zymark Twister Software, click to raise the PlateHandler arm.b. Turn the thumbscrew in the Down direction 10 steps.c. Repeat step 6 until the Plate Handler arm successfully detects the plate.7. Click , then click .If the adjustment was successful, the Plate Handler arm will grasp the plate andremove it from the plate stack.If Plate Handler arm stops before the gripper fingers are able to contact the plateand fails to grasp or pick up the plate, adjust the plate sensor switch:a. Turn the thumbscrew in the Up direction 10 steps.b. Grasp the plate with one hand and, from the Zymark Twister Software,clickto release the plate.c. Replace the reaction plate into input stack 1 of the Plate Handler.8. Repeat steps 6 through 7 until the Plate Handler arm successfully retrieves theplate.9. Grasp the plate with one hand and, from the Zymark Twister Software,click to release the plate.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-39DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument10. Exit the Zymark Twister Software.a. Click .b. Click Quit Application to close the software.A defect in the Zymark Twister Software can cause portions of the program to persistin memory even after the software has been closed. Because the Zymark TwisterSoftware conflicts with the SDS software, the residual elements of the software mustbe closed inside the Windows Task Manager before continuing.1. Confirm that the stack has closed by viewing the Task Manager.a. Press the Crtl + Alt + Del keys in unison.b. In the Windows Security dialog box, click Task Manager.c. In the Task Manager dialog box, confirm that the software has closed bylooking for the Zymark Twister Software entry in the Task list. If thesoftware is still running, click the software entry and click Close to exit theremaining software.d. Select File > Exit.7-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

7900HT FAST Real-Time PCR SystemAligning the Plate HandlerAligning the Plate HandlerWhen to PerformPreparing theInstrument for theAlignmentPerform the following procedure if the Applied Biosystems 7900HT Fast Real-TimePCR System is moved or the Zymark Twister Microplate Handler becomesmisaligned.Symptoms that the Plate Handler is out of alignment include:• Excessive downward movement of the Plate Handler arm (the arm grinds whengrasping or releasing plates)• The Plate Handler arm collides with the plate stacks• The Plate Handler arm releases plates above the bottom of the plate stacks• Reaction plates tip or tilt when placed into the instrument tray by the arm1. Remove the covers for the fixed-position bar code reader and the underlyingplatform.GR20097900HTFront view with RobotGR2009Fixed-position bar code readerand underlying platform covers2. Loosen the three black thumbscrews on the platform connecting the 7900HTinstrument and the Plate Handler base.Black thumbscrews3. Move the instrument tray to the out position.a. Start the Automation Controller Software.If an error dialog box appears reading, ‘Machine calibration values arenot valid. Please refer to documentation for calibration process,’click .b. Click .The 7900HT instrument moves the instrument tray to the out position,perpendicular to the instrument.c. Select File > Exit.The software quits the Automation Controller Software.4. Select > Programs > Zymark Twister Plate Handler > Twister.The Zymark Twister Software starts.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-41DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument5. Click Manual Control to display the Manual Control dialog boxAligningInput Stack 1(ZymarkPosition 4)The alignment of input stack 1 (position 4 in the Zymark Twister Software) is thefirst step in the alignment procedure. This alignment provides the basis for aligningall subsequent stacks on the Plate Handler.1. Place an empty plate into input stack 1(Zymark position 4).2. In the Zymark Twister Software, click position 4.ClickThe Plate Handler arm moves over the input stack.3. Using the Vertical Positioning commands, lower the Plate Handler arm until itis just above the stack.The Vertical Positioning box offers four ways to move the Plate Handler arm:• Move the slider for large increments.• Click inside the slider bar to move the arm in 250 step increments.• Click the lower arrow on the bar to move the arm in 50 step increments.• Click the up or down arrows in the Vertical Adjustment text box to movethe arm in 1-step increments.• Click the Vertical Adjustment text box, enter a value, and press Enter tomove the arm into a specific location.SliderSlider bar (250 steps per click)Down arrow (50 steps per click)Text box arrows (1 step per click)4. Check the rotary position of the Plate Handler arm to confirm that the gripper:• is centered over the stack• will not contact the sides of the stack when lowered7-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Aligning the Plate Handler5. Using the Rotary Adjustment arrows, adjust the rotational position of thegripper so that it is centered over the input stack and will not contact the sideswhen lowered.• To move the Plate Handler arm clockwise, click the up arrow.• To move the Plate Handler arm counter-clockwise, click the down arrow.Up arrow (moves the arm clockwise)Down arrow (moves the arm counter-clockwise)6. Using the Vertical Positioning commands, carefully lower the Plate Handlerarm into the stack. Adjust the Rotary Adjustment value as needed to center thegripper inside the stack.7. After the gripper is centered inside the stack, click .The Plate Handler arm lowers upon the plate.Confirm the following:• the plate is in the middle of the gripper span• the plate sensor switch is contacting the plate• the gripper and plate do not contact the side of the stack8. Click .The gripper grips the plate between its fingers.9. Select .The Plate Handler raises the arm to its highest position. If the plate contacts thesides of the stack, re-adjust the rotary position of the Plate Handler arm until theplate moves freely in the stack.Note: Contact between the plate and the stack or all stacks may be unavoidable.However, try to minimize the contact as much as possible.10. Using the Vertical Positioning commands, raise and lower the Plate Handler armseveral times to check the alignment.11. Lower the Plate Handler arm to the bottom of the plate stack, click ,and click .The software records the rotary position for the Zymark position 4(input stack 1).12. Click .The gripper releases the plate.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-43DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24ABCDEFGHIJKLMNOPABCDEFGHIJKLMNOP1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Chapter 7Maintaining the InstrumentAligning the PlateHandler to theInstrumentThe next step is to align the Plate Handler arm to the instrument tray (Zymarkposition 2). This alignment will ensure a smooth exchange between the Plate Handlerarm and the instrument tray during operation of the instrument.Note: The following procedure requires you to position the Plate Handler relative tothe 7900HT instrument, Before moving the Plate Handler, loosen the three blackthumbscrews on the platform connecting the 7900HT instrument and the PlateHandler.1. If not already present, place an empty plate into input stack 1 (Zymarkposition 4) and pick it up with the Plate Handler arm:a. In the Zymark Twister Software, click position 4.b. Click .c. Click .2. Click position 2.Click hereThe Plate Handler arm moves over the instrument tray.3. Use Vertical Positioning to lower the Plate Handler arm until it is approximately1 cm above the instrument tray.4. Using the Rotary Adjustment arrows, center the gripper and plate along theY-axis of the instrument tray.Center the plate5. Center the gripper and plate along the X-axis of the instrument tray by slidingthe Plate Handler and base towards or away from the 7900HT instrument.Center the plateGR20126. Again, using the software to move the Plate Handler arm, center the gripper andplate along the Y-axis of the instrument tray as explained in step 4.7-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Aligning the Plate Handler7. Using the Vertical Positioning commands, carefully lower the Plate Handlerarm onto the instrument tray and confirm that the plate rests squarely inside it.8. Tighten the three black thumbscrews on the platform connecting the 7900HTinstrument and the Plate Handler.9. Release the plate from the Plate Handler arm.a. Click .b. Click .10. Click .The Plate Handler arm lowers onto the plate.11. Save the rotary and vertical offset information:a. Click , and click .The software records the rotary position for the plate drawer (Zymarkposition 2).b. Click , and click .The software records the vertical position for the plate drawer.Re-checking theInput Stack 1Now that the positions of the Plate Handler and instrument are fixed, the PlateHandler stacks can be aligned and the positional values recorded.1. Place an empty plate into input stack 1.2. In the Zymark Twister Software, click the icon for stack 4.The Plate Handler arm moves over the input stack.3. Using the Vertical Positioning commands, lower the Plate Handler arm until itis 1 cm above the stack and verify that it is centered on the stack. If necessary,center the stack using the Rotary Adjustment arrows.4. Carefully lower the Plate Handler arm into the stack. Center the gripper as itmoves down the stack by adjusting the Rotary Adjustment arrows if needed.5. After the Plate Handler arm is centered inside the stack, click .The Plate Handler arm lowers upon the plate.Confirm the following:• The plate is in the middle of the gripper span.• The plate sensor switch is contacting the plate.• The gripper does not contact the side of the stack.6. Click .7. Click .The Plate Handler raises the arm to its highest position. If the plate contacts thesides of the stack, re-adjust the rotary position of the Plate Handler arm until theplate moves freely inside the stack.Note: Contact between the plate and the stack or all stacks may be unavoidable.However, try to minimize the contact as much as possible.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-45DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument8. Click , and click .The software re-records the rotary position for the input stack 1 (Zymarkposition 4).9. While holding the plate, click and remove the plate.Defining theBottom of theStackThe Automation Controller Software requires a bottom position value for all stacks.This value is used to prevent the Plate Handler arm from colliding or grinding as itmoves to the bottom of each stack.1. Remove all plates from the instrument and the Plate Handler arm.2. Place an empty plate into the output stack (Zymark position 0).3. In the Zymark Twister Software, click position 0.The Plate Handler arm moves over the output stack.4. Using the Vertical Positioning commands, lower the Plate Handler arm until itis just above the stack.5. Check the rotary position of the Plate Handler arm to confirm that the gripper:• is centered over the stack• will not contact the sides of the stack when lowered6. Using the Rotary Adjustment arrows, adjust the rotational position of thegripper so that it is centered over the input stack and will not contact the sideswhen lowered.7. Using the Vertical Positioning commands, carefully lower the Plate Handlerarm into the stack. Adjust the Rotary Adjustment value as needed to center thegripper inside the stack.8. After the gripper is centered inside the stack, click .The Plate Handler arm lowers upon the plate.Confirm the following:• The plate is in the middle of the gripper span• The plate sensor switch is contacting the plate• The gripper does not contact the side of the stack9. Click .The gripper grips the plate between its fingers.10. Select .The Plate Handler raises the arm to its highest position. If the plate contacts thesides of the stack, re-adjust the rotary position of the Plate Handler arm until theplate moves freely in the stack.Note: Contact between the plate and the stack may be unavoidable. However,try to minimize the contact as much as possible.11. Using the Vertical Positioning commands, raise and lower Plate Handler armseveral times to check the alignment.12. Lower the Plate Handler arm and click , and click .The software records the rotary position for position 0 (the output stack).7-46 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Aligning the Plate Handler13. Click the .14. While holding the plate, click and remove the plate.15. In the Vertical Adjustment field, enter –3200, and press Enter. The PlateHandler lowers the arm to a position near the base of the output stack.16. Carefully lower the Plate Handler arm until it is approximately 1–2 mm from thebottom of the stack.17. Click , click , and record the number in the VerticalAdjustment field. The software records the vertical position for position 0 (theoutput stack).18. Click . The Plate Handler raises the Plate Handler arm to its highestposition.19. In the Vertical Adjustment field, enter the Vertical Offset value determined instep 17, and press Enter. The Plate Handler lowers the Plate Handler arm to aVertical Offset position.20. If necessary, readjust the Vertical Offset value and repeat steps 18 through 19until satisfied with the setting.Defining thePositions of theRemainingStacks1. Place an empty plate into input stack 2 (Zymark position 5).2. In the Zymark Twister Software, click position 5.The Plate Handler arm moves over the input stack.3. Using the Vertical Positioning commands, lower the Plate Handler arm until itis approximately 1 cm above the stack and center it using the RotaryAdjustment arrows.4. Carefully lower the Plate Handler arm into the stack. Center the gripper as itmoves down the stack by adjusting the Rotary Adjustment arrows as needed.5. After the Plate Handler arm is centered inside the stack, click .The Plate Handler arm lowers upon the plate.Confirm the following:• The plate is in the middle of the gripper span.• The plate sensor switch is contacting the plate.• The gripper does not contact the side of the stack.6. Click .7. Click .The Plate Handler arm raises to its highest position. If the plate contacts thesides of the stack, re-adjust the rotary position of the Plate Handler arm until theplate moves freely in the stack.Note: Contact between the plate and the stack may be unavoidable. However,try to minimize the contact as much as possible.8. Using the Vertical Positioning commands, raise and lower Plate Handler armseveral times to check the alignment.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-47DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

7900HT FAST Real-Time PCR SystemChapter 7Maintaining the Instrument9. Click , and click .The software records the rotary position for the Zymark position 5 (inputstack 2).10. Repeat steps 1 through 9 for input stacks 3 and 4 to define Rotary Offset valuesfor the remaining positions 6 and 7:Zymark position 7(input stack 4)Zymark position 6(input stack 3)11. Exit the Zymark Twister Software.a. Click .b. Click Exit.The software closes.A bug in the Zymark Twister Software can cause portions of the program topersist in memory even after the software has been closed. Because the ZymarkTwister Software conflicts with the SDS software, these residual elements mustbe closed inside the Windows Task Manager before continuing.12. Confirm that the Zymark Twister Software has closed by viewing the TaskManager.a. Press the Ctrl + Alt + Del keys in unison.b. In the Windows Security dialog box, click Task Manager.c. In the Task Manager dialog box, confirm that the Twister software hasclosed by looking for the Twister software entry in the Task list. If thesoftware is still running, click the software entry and click End Task toexit the software.d. Select File > Exit to quit the Task Manager.13. Replace the covers for the fixed-position bar code reader and the underlyingplatform (removed in step 1 on page 7-41).GR20097900HTFront view with RobotGR2009Fixed-position bar code readerand underlying platform covers7-48 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

7900HT FAST Real-Time PCR SystemAligning the Fixed-Position Bar Code ReaderAligning the Fixed-Position Bar Code ReaderDescriptionPreparing theInstrument for theAlignmentThe fixed-position bar code reader must be set so that it automatically scans theplate’s bar code as the plate is placed into the instrument tray by the Plate Handler.1. Remove the cover for the fixed-position bar code reader.GR20097900HTFront view with RobotGR2009Fixed-position bar code readercover2. Power on the computer.3. Start the Automation Controller Software.4. Click .The 7900HT instrument moves the instrument tray to the out position,perpendicular to the instrument.5. Select File > Exit.The software quits the Automation Controller Software.Positioning theFixed-PositionBar Code ReaderIMPORTANT! The instrument tray must be in the OUT position to align the bar codereader.1. Place a plate with bar code onto the instrument tray.IMPORTANT! Orient the plate so that well A1 aligns to the A1 position of theinstrument tray and that the bar code faces the fixed-position bar code reader.Well position A1GR2018Bar code2. Select > Programs > PSC Laser Data > LDHOST.The LDHOST software starts and displays the LDHOST window.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-49DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument3. Establish communication with the fixed-position bar code reader:a. Click (Edit).b. Click (Terminal).The software opens the Edit Configuration and Terminal dialog boxes.c. In the Device Control dialog box, click (Connect to Device).The terminal window displays the fixed-position bar code reader response.d. Click to close the Information dialog box.The LD Host program communicates with the bar code reader and updates theEdit Configuration dialog box with the current configuration settings.4. Configure the software for the alignment:a. In the bottom of the Edit Configuration dialog box, locate and select theOp. Modes tab.Op. Modes tabNote: You may need to use the arrows located in the bottom of the dialog box tolocate the Op. Modes tab.b. In the Operating modes selection group of the Edit Configuration dialogbox, click the arrow to the right of the Mode heading and select Test fromthe drop-down list.Mode drop-down listc. In the Device Control dialog box, click RAM to toggle to EEPROMmode.RAM buttond. In the Device Control dialog box, click Send.e. In the Confirm dialog box, click YES to save to EEPROM.The fixed-position bar code reader begins a continuous repeating scan of the barcode. The software updates the Terminal dialog box every 0.5 sec indicating thepercentage of accurate reads completed during the 0.5 sec interval.7-50 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Aligning the Fixed-Position Bar Code Reader5. Loosen the black positional adjustment knob on the fixed-position bar codereader, and position the scan head of the reader as far as possible from the platewhile maintaining the orientation towards the bar code on the plate (see below)Scan head of the fixed-positionbar code readerGR2018Black positionaladjustment knob6. While watching the Terminal dialog box, slowly adjust the orientation of thefixed-position bar code reader until the percent successful reading displays thehighest number possible.Percent successful readsNote: It may be helpful to briefly place a sheet of white paper in front of theplate bar code to view the area scanned by the laser.7. When satisfied with the alignment, tighten the black positional adjustment knobon the fixed-position bar code reader.8. Restore the fixed-position bar code reader to normal operation:a. In the Edit Configuration dialog box, change from Test back to Serial onLine.Mode drop-down listb. In the Device Control dialog box, confirm that EEPROM is still selected,and click Send.c. In the New Decision dialog box, click YES to save to EEPROM.The bar code reader stops scanning the plate bar code and resumes normaloperation.9. Click Exit ( ) to quit the LDHOST window.The LDHOST window closes.10. Replace the cover for the fixed-position bar code reader (from step 1 onpage 7-49).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-51DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentCleaning and Replacing Gripper Finger PadsWhen to PerformThe adhesive used to affix bar code labels to certain brands of microplates can buildup on the gripper pads of the Zymark Twister Microplate Handler. Over time, theresidue can cause the gripper pads to stick to the microplates while handling them,causing misfeeds. To prevent buildup, inspect the gripper pads monthly and clean orreplace the pads as needed.MaterialsRequiredTable 7-3MaterialMaterials Required for Replacing the Gripper Finger PadsPart NumberFinger Pad Replacement Kit, containing 10 finger pads 4315472Flat-blade screwdriver, small —Phillips head screwdriver, small —Isopropanol in a squeeze bottle —CHEMICAL HAZARD. Isopropanol is a flammable liquid andvapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged orrepeated contact may dry skin and cause irritation. It may cause central nervoussystem effects such as drowsiness, dizziness, and headache, etc. Please read theMSDS, and follow the handling instructions. Wear appropriate protective eyewear,clothing, and gloves.Cleaning theFinger PadsReplacing theFinger PadsTo clean the finger pads, wipe each pad thoroughly with Isopropanol until the residuehas been resolved. If the pads appear rough or the adhesive cannot be removed,replace the pads as described below.1. Using the Phillips-head screwdriver, remove the two small Phillips-head screwsfrom the fingers on each side of the gripper, then remove the fingers.Note: Move the Plate Handler arm into any position where it is easy to accessthe screws.2. Using a small flat-blade screwdriver, pry the worn finger pads off the fingers.Note: The manufacturer recommends replacing all finger pads at the sametime.3. Clean any residual adhesive off the fingers using isopropanol.4. Remove a replacement finger pad from the paper backing, and place the fingerpad on the appropriate finger position.5. Repeat for the remaining finger pads.6. Install the fingers with the fingers pointing down and the finger pads toward thecenter of the gripper.7. Insert the screws into the fingers and tighten.Note: The screws do not automatically align the grippers. Make sure that thefinger pads are making good contact with the plate when the arm grips a plate.7-52 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Section 7.3 Maintaining the Computer and SoftwareSection 7.3Maintaining the Computer and SoftwareIn This SectionThis section contains the following information:General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54Maintaining the SDS software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55Note: The SDS software is a multicomponent system that must be maintained toensure optimal operation of the Applied Biosystems 7900HT Fast Real-Time PCRSystem. Although an Applied Biosystems service engineer will complete most of themaintenance, this section discusses important issues that you should understand.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-53DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the InstrumentGeneral Computer MaintenanceMaintenanceScheduleThe computer connected to the 7900HT instrument requires regular maintenance toensure reliable operation of the Applied Biosystems 7900HT Fast Real-Time PCRSystem components. Applied Biosystems recommends the following tasks as part ofroutine maintenance of the computer system:Table 7-4Maintenance Schedule for the Instrument ComputerMaintenance TaskArchive or Remove Old SDS FilesDefragmenting the Hard DriveUpgrading the Operating System SoftwareUpgrading the SDS SoftwarePerformWeeklyMonthly or beforefragmentation reaches 10%When available/advisableWhen availableDeveloping a Data Management StrategyApplied Biosystems recommends developing a strategy for dealing with the filesproduced by the SDS software. During a single day of real-time operation, theApplied Biosystems 7900HT Fast Real-Time PCR System can generate over 200 MBof data. Without a strategy for distributing and archiving SDS-related files, the7900HT instrument can easily fill the hard drive of the computer within just a fewweeks of operation. See “Managing Sequence Detection System Data” on page 1-17for a discussion of management strategies.ArchivingSDS FilesDefragmentingthe Hard DriveTo conserve space on the computer hard drive, SDS files can be archived using a datacompression utility. The compression utility archives files by encoding them in acompressed form, thereby reducing the size of a file. SDS files can be compressedand decompressed many times.Several commercially available compression utilities are available. PKZIP and *.arcare archive formats common to the Microsoft Windows operating system.Applied Biosystems recommends defragmenting the hard drive of the computerattached to the instrument at least once every week or before fragmentation reaches10%. As the Applied Biosystems 7900HT Fast Real-Time PCR System is used andfiles are deleted and created, the free space on the computer hard drive eventually issplit into increasingly smaller blocks (called “clusters”). Consequently, as the SDSsoftware creates new files and extends old ones, the computer cannot store each filein a single block. Instead, the system will ‘fragment’ the files by scattering theircomponent pieces across different sectors of the hard drive.The fragmentation of SDS files decreases the performance of both the SDS softwareand the computer operating system. As the hard drive becomes fragmented, programstake greater time to access files because they must perform multiple seek operationsto access the fragments.7-54 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Maintaining the SDS softwareSeveral commercially available software utilities are available for repairingfragmented file systems. The software utility defragments broken files by combiningtheir component pieces at a single location on the hard drive, thereby optimizingsystem performance.Upgrading theOperatingSystem SoftwareDo not upgrade the operating system of the computer connected to the 7900HTinstrument unless instructed to do otherwise by an Applied Biosystemsrepresentative. New versions of the Microsoft Windows operating system can beincompatible with the SDS software and render it and the instrument inoperable.Applied Biosystems service engineers maintain the operating system software as partof planned maintenance visits. During the visit, an engineer will update the computeroperating system as upgrades become available and are validated by AppliedBiosystems.Maintaining the SDS softwareUser AccessRequirementUpgrading theSDS SoftwareReinstalling theSoftwareIMPORTANT! You must have administrator privileges on the computer to installand/or upgrade the SDS software.Applied Biosystems continually develops the SDS software to provide increasedfunctionality and reliability of the Applied Biosystems 7900HT Fast Real-Time PCRSystem. As updates become available, Applied Biosystems sends notifications of theupgrades to all Applied Biosystems 7900HT Fast Real-Time PCR System customers.If an upgrade is user-installable, it can be found on the Applied Biosystems CompanyWeb site (see “How to Obtain Services and Support” on page xii).Note: Applied Biosystems service engineers perform regular software updatesduring planned maintenance visits.On rare occasions, when a piece of the SDS software becomes corrupt, it may benecessary to re-install the software. In the event that the software must bere-installed, observe the following guidelines to re-install or upgrade the software.• Unless instructed to do otherwise, remove the SDS software using the uninstallutility. Do not delete the program subdirectory from the Program Files directory.• Install the SDS software under a user login that has administrator privileges onthe computer.• Unless instructed to do otherwise, re-install the SDS software to the samedirectory as the previous installation.• Review all documentation accompanying the new software (such as installationnotes or user bulletin). The updated version of the software may contain newfeatures that require special consideration.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-55DRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Chapter 7Maintaining the Instrument7-56 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Maintenance.fm

Troubleshooting 88In This Chapter Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9Pure Dye Runs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11Real-Time Runs (Quantitative PCR and Dissociation Curves). . . . . . . . . . . . . . . 8-12End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . 8-17TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-1DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingTroubleshooting TableOverviewThe following table is designed to help you troubleshoot most of the problems youmay encounter while using the Applied Biosystems 7900HT Fast Real-Time PCRSystem.The information in the table is arranged by category as follows:• Chemistry problems• Run problems• Instrument and Automation Accessory ProblemsEach category contains subcategories, followed by a brief description of thesymptoms you might encounter.To use this table, look for the category and the symptom you are experiencing. Thepage number in the right-hand column corresponds to a description of the possiblecause(s) and recommended action(s) for that particular problem.Table 8-1Troubleshooting TableCategory Symptom PageChemistry and Run ProblemsChemistry Low Precision 8-5IrreproducibilitySoftware InstallationSDS SoftwareRun ProblemsUnable to finish installation(Install program appears to b e frozen)Installation Interrupted8-5Background Runs Software will not extract background data 8-9Background is too high (greater than 2500)Pure Dye Runs Software will not extract pure dye data 8-11Raw data from pure dye run appears strangeSignals plateau (saturation)Signal is too low (< 10,000 FSU)More than two outliers per dye in a single rowReal-Time Runs (Quantitative PCR and Dissociation Curves) 8-12End-Point Runs (Allelic Discrimination) 8-138-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Troubleshooting TableTable 8-1Troubleshooting TableCategory Symptom PageInstrument and Automation Accessory ProblemsSoftware and 7900HTInstrumentZymark Twister MicroplateHandler andFixed-Position Bar CodeReaderSDS software will not startSoftware crashes/freezes the computer ordisplays an error messageCommunication errorThermal cycler errorsAutomation Controller Software cannot find aplate document fileComputer and/or software displays the RunCompleted Successfully dialog box but will notrespond and appears to be frozenRun will not startComputer is slow when analyzing data,opening or closing dialog boxes, and othersoftware processes.The computer will not logon to the WindowsOperating System.The computer will not boot up at all.Plate Handler emits grinding noise whenpicking up or putting down platesPlate Handler arm contacts racks whenretrieving or stacking platesPlate Handler arm releases plates awkwardlyinto the plate stackReaction plates tip or tilt when placed into theinstrument tray by the Plate Handler armPlate Handler fails to sense or grasp platesPlates stick to the gripper fingers of the PlateHandler armPlate Handler does not restack plates inoriginal locationsFixed-position bar code reader not readingplate bar codes8-148-17Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-3DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingTable 8-1Troubleshooting TableCategory Symptom PageTaqMan ® Low DensityArraySDS Enterprise DatabaseAfter removing the Low Density Array from itspackaging, the fill consumable is damaged(creased, bent, or folded).After removing the Low Density Array from itspackaging, the aluminum foil backing isdamaged (creased, bent, or folded).After removing the Low Density Array from itspackaging, dust or other particulates settle onthe reaction wells (optical side of the LowDensity Array).After removing the Low Density Array from itspackaging, water condenses on the reactionwells (optical side of the Low Density Array).After pipetting, too little PCR reaction mixturehas gone into the fill reservoir.After pipetting, some of the PCR reactionmixture leaks out of the vent port in the fillreservoir.The PCR reaction mixture leaks during PCRcycling.After a mean assay result and standarddeviation of the replicates are calculated, aspecific replicate appears to be an outlier.Archive or restore operation was terminatedprematurelyCannot Connect to the Database8-188-208-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Low Precision or IrreproducibilityLow Precision or IrreproducibilityOverviewImproperThreshold SettingThere are many reasons why an assay run with the Applied Biosystems 7900HT FastReal-Time PCR System can have less than optimal precision. Factors that can affectprecision are described in detail below.Improper Threshold Setting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5Imprecise Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6Non-Optimized Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6Incomplete Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6Splashing PCR Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6Drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7Writing on the Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7Fluorescent Contamination on the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7Errors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7Contaminated Sample Block. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7Improper or Damaged Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8Low Copy Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8Use of Non-Applied Biosystems PCR Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . 8-8The key to high-precision quantitative PCR is accurate detection of the geometricphase. The Applied Biosystems 7900HT Fast Real-Time PCR System typicallydelivers sufficient sensitivity so that at least three cycles of the geometric phase arevisible, assuming reasonably optimized PCR conditions. The SDS softwarecalculates a fixed signal intensity, called a threshold, that each signal generated fromPCR amplification must reach before it is recognized by the software as actualamplification. The calculated threshold is an approximation, and should be examinedand modified as needed.Modifying the ThresholdIn a real-time document of the SDS software, the threshold can be modified via theAmplification plot view following analysis of the run data. See “Setting the Baselineand Threshold Values” on page 6-46 for more information.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-5DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingImprecisePipettingThe calculated quantities of target nucleic acid are directly affected by how preciselythe template volumes are added to the reaction mixes. Other individually addedreagents are also affected by pipetting precision (such as, variable magnesium affectsamplification efficiency).Using Master MixesFor this reason, Applied Biosystems highly recommends using a master mix. Allcommon components to a set of reactions should be mixed together and thendispensed to the wells of the plate. Sub-master mixes can be used to further improvethe precision of identical replicates. For example, instead of pipetting 5 µL of thesame template into four replicate wells, pipette 20 µL of the template into asub-master mix, then divide the sub-master mix into four equal parts foramplification. When making each master mix, add 5–10% additional volume tocompensate for pipetting losses.Using PipettorsPipetting precision is also improved by:• Calibrating and servicing the pipettors regularly• Pipetting larger volumes• Reducing the number of pipetting steps whenever possible• Increasing the consistency of the pipetting methodConsult the manufacturer about the correct method of dispensing liquid volumesaccurately from the pipettor. For example, some pipettors are designed to deliver thedesignated volume at the first plunger stop, so ‘blowing out’ the residue may causeerror. Also, before using a new pipettor tip to serially dispense a master mix, wet thetip once by drawing up some of the master mix and dispensing it back into the mixagain.Non-OptimizedChemistryIncompleteMixingAir BubblesSplashing PCRReagentsChemistries that have not been optimized may be susceptible to inconsistencies. Tomaximize precision and reaction efficiency, optimize the primer and probeconcentrations of each individual assay used. Refer to the TaqMan Universal PCRMaster Mix Protocol (PN 4304449) for specific information about optimizing probeand primer concentrations for TaqMan-related chemistries.For maximum precision, the PCR master mix must be mixed to uniformity. After allreaction components are added to master mix, it should be vortexed for 4–5 secondsbefore aliquoting it to the wells of the plate. Any dilutions performed during theassay should also be vortexed.Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, thereagents would be applied to the wells using a pipetting technique that does not formair bubbles. However, if a plate does contain air bubbles, they can usually be removedby swinging, tapping, or briefly centrifuging the reaction plate.If PCR reagents splash the undersides of the optical adhesive covers, the heat fromthe lid may bake the liquid to the cover and may distort the signal. If splashingoccurs, briefly centrifuge the reaction plate to remove all traces of liquid from thecaps.8-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Low Precision or IrreproducibilityDropsWriting on theReaction PlatesFluorescentContamination onthe PlatesErrorsContaminatedSample BlockDrops of reagents that cling to the sides of the wells may not contact the thermalcycler sample block and consequently may not amplify. If the drop slides into the mixduring PCR, then the amplified products will become diluted and the result will beless than replicate wells that did not have drops. Therefore, carefully monitor thereaction plate as it is being transferred into the thermal cycler or 7900HT instrument.If you observe any drops, take steps to remove them, such as centrifugation.Do not write on any surface of the optical plates or the optical adhesive covers. Thefluorescent properties of the ink can potentially affect the fluorescence emissionfrom the plate and alter the results. Instead, note the contents of each well on a sheetof paper, or on a printout of the sample setup.Many compounds found in laboratories are fluorescent. If they come into contactcertain optical surfaces, such as the optical adhesive covers, the fluorescent resultsmay be affected. For example, it has been noted that the powder used to lubricate theinsides of plastic gloves often contains fluorescent compounds. Use only powder-freegloves and do not needlessly touch the reaction plates or optical adhesive seals.Human errors from time to time are inevitable, such as pipetting into the wrong well,or making a dilution mistake.Human error can be reduced in the following ways:• Perform the assay in a systematic fashion. For example, the pattern of samplepositions should be simple (avoid putting gaps in the rows).• When pipetting the master mix, look directly down into the reaction plate so thatyou can verify the transfer of the solution.• If adding a small-volume reagent, such as template, place the drop of liquid onthe side of the well. Briefly tap or centrifuge the plate afterwards to bring thedroplet down into the well.• After all pipetting is complete, visually inspect all the wells to confirm thepresence of the reagent drops. Tapping or centrifuging the reaction plate willcause all the drops to slide down into the wells simultaneously.• When making serial dilutions, be sure to change the pipet tip after each dilutionstep.• Visually inspect the liquid volumes being pipetted to verify that the volume isapproximately correct. A common mistake is using the wrong pipettor volumesetting (such as setting 20 µL instead of 2.0 µL).• Visually inspect the volumes of the completed reactions, looking for any wellsthat have volumes that do not match those of the other wells.Any material contaminating the sample block can affect the results. For example,mineral oil reduces thermal transfer. Residue from writing on reaction plates darkensthe wells, absorbing light.The sample blocks should be periodically inspected for cleanliness. Sample blockcontamination can be visualized by running a background plate and inspecting theresulting background signal for aberrant peaks above 2500 FSU (see page 7-16). Seepage 7-14 for instructions on decontaminating the sample block.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-7DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingImproper orDamagedPlasticsLow CopyTemplatesUse ofNon-AppliedBiosystems PCRReagentsOnly Applied Biosystems optical plates, optical adhesive covers, and optical flat capsshould be used with the Applied Biosystems 7900HT Fast Real-Time PCR System.The plastics that comprise the optical parts undergo special testing for the absence offluorescent impurities. Optical plates are frosted to improve the degree and precisionof light reflection. Bent, creased, or damaged plastics may adversely affect thetransmission of fluorescent signal or prevent proper sealing of a well resulting inevaporation, change in sample volume, and altered PCR chemistry. Make sure to usethe correct plastics and visually inspect each reaction plate before use.Note: See Appendix C, “Kits, Reagents, and Consumables,” for a list of compatibleconsumables and reagents.When amplifying samples that contain very low quantities of nucleic acid (generallyless than 100 molecules), expect lowered precision due to the Poisson distributionand biochemical effects related to binding probabilities. Low copy templates are alsomore susceptible to losses due to non-specific adhesion to plastic wells, pipettor tips,etc. The addition of carrier to the sample, such as yeast tRNA or glycogen, can helpprevent these losses, increasing the precision and sensitivity of the assay.The Applied Biosystems buffer contains an internal passive reference molecule(ROX dye), which acts as a normalization factor for fluorescent emissions detectedin the samples.IMPORTANT! Non-Applied Biosystems PCR buffers may not contain the ROXpassive reference dye. If running non-Applied Biosystems chemistry, be sure to setthe passive reference for your experiment as explained on “Setting the PassiveReference” on page 3-18.8-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Background RunsBackground RunsBackgroundTroubleshootingTableTable 8-2Troubleshooting Background RunsObservation Possible Cause Recommended ActionSoftware will not extractbackground dataBackground is too high(=>2500)During setup, the wrongplate type was assigned tothe plate documentBackground is too high(≥ 2500 FSU a )Sample blockcontaminationBackground platecontaminationRun a new backgroundplate document with theproper plate type setting.See below.1. Construct and run a newbackground plate.2. See “IsolatingSample BlockContamination” below.aFluorescent standard units – The measure of amplitude displayed along the Y-axis of theBackground Plot.IsolatingSample BlockContaminationSignals exceeding 2500 FSU are considered outside the limit of normal backgroundfluorescence and indicates that the either the background plate or the sample blockmodule may be contaminated.1. If not already open, open the plate document for the background run.2. In the toolbar, click (Hide/Show System Raw Data Pane).The SDS software displays the raw data pane for the background run.3. Select all wells in the plate document.4. Inspect the raw background data for an aberrant spectral peak or peaks.Wells producing raw spectra that exceed 2500 FSU are considered irregular andcould be contaminated. The following figure illustrates the raw data producedby a run on a sample block module containing a contaminated well.Possible contaminationApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-9DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8Troubleshooting5. Identify the location(s) of the contaminated well(s) on the sample block byselecting increasingly smaller regions of the plate document (see below).a. The raw data fromthe selected wellscontains the peak.The contaminatedwell must be incolumns 7-12.b. The raw data fromthe selected wellsdoes not containthe peak.The contaminatedwell must be in thelast four wells ofcolumns 7-12.c. The raw data fromthe selected wellscontains the peak.The contaminatedwell must be in thelast four wells ofcolumns 10-12.d. The raw data fromthe selected wellscontains the peak.The contaminatedwell must be in thelast two wells ofcolumns 10-12.e. By selecting eachof the wells fromthe last two wellsof columns 10-12,the location of thecontaminatedwell (G10) isdetermined.8-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Pure Dye Runs6. Repeat step 4 until you identify the location of each contaminated well.7. Decontaminate the sample block as explained in “Decontaminating the SampleBlock” on page 7-14.8. Run a background plate to confirm that the contaminants have been removed.If the contamination is present after running the background plate for a secondtime, the background plate is likely to be the source of contamination.Pure Dye RunsPure DyeTroubleshootingTableTable 8-3Troubleshooting Pure Dye RunsObservation Possible Cause Recommended ActionSoftware will not extractpure dye dataRaw data from pure dyerun appears strange(see below)During plate setup, thewrong plate type wasassigned to the platedocumentA background plate was notrun before the pure dyeplate or cardPure dye plate or card wasloaded backwardsCreate and run a new puredye plate document with theproper plate type settingRun a background plate,then run the pure dye plateor card again1. Verify the pure dyewavelengths are asexpected.2. Rerun the pure dye plate.or cardSignals plateau (saturation) Intensity is set too high/low Call Applied BiosystemsTechnical Support.Signal is too low(< 10,000 FSU)More than two outliers perdye in a single row• Evaporation• ContaminationRerun the pure dye plate orcard. If the problempersists, discard the puredye plate or card and run anew one.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-11DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingReal-Time Runs (Quantitative PCR and Dissociation Curves)TroubleshootingAnalyzed DataRaw Data PlotWhen faced with irregular data, you can use the SDS software to diagnose somechemistry- and instrument-related problems. The following table contains asummary of checks for verifying the integrity of your run data and to help you begintroubleshooting potential problems.The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) forthe selected wells during each cycle of the real-time PCR.What to look for:• Signal tightness and uniformity – Do the raw spectra signals from replicategroups and controls exhibit similar spectral ‘profiles’? If not, the plate orsample block could be contaminated.• Characteristic signal shape – Do the samples peak at the expectedwavelengths? For example, samples containing only FAM dye-labeledTaqMan ® probes should not produce raw fluorescence in the wavelength of aVIC ® dye component. A signal present in wells that do not contain the dye couldindicate that the sample, master mix, or well contains contaminants.• Characteristic signal growth – As you drag the bar through the PCR cycles, doyou observe growth as expected? Absent growth curves may indicate a pipettingerror (well lacks template).• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturationcan be an indication that a well contains too much template or fluorescent signal.MulticomponentPlotThe Multicomponent Plot displays a plot of normalized multicomponent data from asingle well of a real-time run. The plot displays the component dye signals thatcontribute to the composite signal for the well.What to look for:• Correct dyes displayed – Does the plot display all dyes as expected? Thepresence of an unexpected dye may be the result of an error in detector setup,such as assigning the wrong reporter or quencher dye.• ROX dye fluorescence level – Does the ROX dye signal fluoresce below thereporter dyes? If not, the lack of reporter fluorescence may be caused by anabsence of probe in the well (a pipetting error).• Background fluorescence – Do all dyes fluoresce above the background? TheBackground signal is a measure of ambient fluorescence. If a dye fails to fluoresceabove the background, it is a strong indication that the well is missing probeslabeled with the dye (well does not contain probe, PCR master mix, or both).• MSE Level – The MSE (mean squared error) is a mathematical representationof how accurately the multicomponented data fits the raw data. The higher theMSE value, the greater the deviation between the multicomponented data andthe raw data.8-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

End-Point Runs (Allelic Discrimination)Amplification PlotThe Amplification plot displays data from real-time runs after signal normalizationand Multicomponent analysis. It contains the tools for setting the baseline andthreshold cycle (C T ) values for the run.What to look for:• Correct baseline and threshold settings – Are the baseline and thresholdvalues set correctly?Identify the components of the amplification curve and set the baseline so thatthe amplification curve growth begins at a cycle number that is greater than thehighest baseline number.IMPORTANT! Do not adjust the default baseline if the amplification curvegrowth begins after cycle 15.Identify the components of the amplification curve and set the threshold so that it is:• Above the background• Below the plateaued and linear regions• Within in the geometric phase of the amplification curve• Irregular amplification – Do all samples appear to have amplified normally? Thethree phases of the amplification curve should be clearly visible in each signal.• Outlying amplification – When the run data is viewed in the C T vs. WellPosition plot, do replicate wells amplify comparably? Wells producing C Tvalues that differ significantly from the average for the associated replicatewells may be considered outliers.If a plate produces non-uniformity between replicates, some samples on the platecould have evaporated. Check the seal of the optical adhesive cover for leaks.End-Point Runs (Allelic Discrimination)TroubleshootingAnalyzed DataRaw DataWhen faced with irregular data, you can use the SDS software to diagnose somechemistry- and instrument-related problems. The following table contains asummary of checks for verifying the integrity of your run data and to help you begintroubleshooting potential problems.The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) forthe selected wells during each cycle of the PCR.What to look for...• Signal tightness and uniformity – Do the raw spectra signals from replicategroups and controls exhibit similar spectral ‘profiles’? If not, the plate orsample block could be contaminated.• Characteristic signal shape – Do the samples peak at the expectedwavelengths? For example, samples containing only FAM dye-labeled TaqManprobes should not produce raw fluorescence in the peak wavelength of the VICdye component. A signal present in wells that do not contain the dye couldindicate that the sample, master mix, or well contains contaminants.• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturationcan be an indication that a well contains too much template or fluorescent signal.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-13DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingSoftware and 7900HT InstrumentTroubleshootingSoftware andComputerProblemsTable 8-4Troubleshooting Software and Computer ProblemsObservation Possible Cause Recommended ActionUnable to finish installation(Install program appears tobe frozen)Installer dialog box ishidden from view1. Press and hold Alt.2. Press Tab until the Installericon is selected.3. Release both keys.The installer dialog box returnsto the front of the desktop.SDS Software version 2.2does not function after theinstallation of SDS Softwareversion 2.2.1SDS software will not startThe software crashes/freezesthe computer or displays anerror messageSDS Software version2.2 is automaticallyremoved during theinstallation• Incorrect start-upsequence• Corrupted software• Computerhardware failure• Operating System(OS) corruption• Loose bar codereader cableReinstall the SDS Softwareversion 2.2Follow the solutions listed untilthe symptom goes away.1. Power off the 7900HTinstrument.2. Check cable connections.3. Restart the computer andlogon to the computer.4. Power on the 7900HTinstrument.5. Start the SDS software.6. Restart the computer andlogon to your computer.7. Reinstall the SDS software.8. Start the SDS software.123Contact Applied BiosystemsService for OS problems or ifthe computer will not boot up atall. You may have to reload theOS from the CDs.4Contact Dell for troubleshootingthe computer hardware.8-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Software and 7900HT InstrumentTable 8-4Troubleshooting Software and Computer ProblemsObservation Possible Cause Recommended ActionCommunication errorThermal cycler errorsAutomation ControllerSoftware cannot find a platedocument fileDialog box does not respondto mouse clicks or keystrokesCables are connectedincorrectlySample block modulenot fully engagedFile not in correctlocationJava Runtime ErrorCheck cable connections andCOM port setup. See“Instrument Connections” onpage 1-10.Reseat the sample blockmodule as explained“Replacing the Sample Block”on page 7-6.Remove file entry from platequeue and add the file to theplate queue again.Click the close box of the dialogbox to close it.Run will not start No calibration file Perform background and puredye runs.No background data inSee “Performing a Backgroundcalibration fileRun” on page 7-16 and(background run has“Performing a Pure Dye Run”not been performed)on page 7-20.No pure dye data incalibration file(pure dye run has notbeen performed)Calibration file doesnot contain pure dyedata for a dye used onthe plate documentCalibration file wascreated on anotherinstrumentDisk drive containingthe plate documenthas less than 50 MB offree spaceHeated cover cannotreach runningtemperature becauseno plate loadedCheck the capacity of thedestination drive. If less than50 MB of free space remains,remove or archive existing datafiles (see page 7-54).Open the instrument tray andcheck that the instrumentcontains a plate.Instrument traycontains a plateOutput stack containsa plate or platesRemove all plates from theoutput stack of the PlateHandler before starting thequeue.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-15DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingTable 8-4Troubleshooting Software and Computer ProblemsObservation Possible Cause Recommended ActionComputer is slow whenanalyzing data, opening orclosing dialog boxes, andother software processesThe computer will not logonto the Windows OperatingSystemThe computer will not bootup at allHard drive isfragmentedHard drive is almostfullLogon window doesnot appearYou are not logged onas the AdministratorAfter the abovesolutions have beentried, the problem isstill not fixedCables are notconnected or are notseated properlyAfter the abovesolution has beentried, the problem isstill not fixedDefragment the hard drive asexplained on “Defragmentingthe Hard Drive” on page 7-54.Remove or archive existingdata files as explained on“Archiving SDS Files” onpage 7-54.Restart the computer and logonto your computer.1. Logoff of your computer.2. Logon again as theAdministrator.Contact Dell for troubleshootingthe computer hardware or OS.Check the cables.The boot disk is corrupted.1. Boot directly off of theWindows NT ® OperatingSystem Installation CD.2. Boot off of the emergencydisk.3. Reload the Windows NTOperating System from theCD.Contact Dell for troubleshootingthe computer hardware.8-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Zymark Twister Microplate Handler and Fixed-Position Bar Code ReaderZymark Twister Microplate Handler and Fixed-Position BarCode ReaderAutomationAccessoryTroubleshootingTableTable 8-5Troubleshooting the Automation AccessoryObservation Possible Cause Recommended ActionPlate Handler emitsgrinding noise whenpicking up or putting downplatesVertical offset too lowPlate detector switch settoo highRe-align the Plate Handleras explained in “Aligning thePlate Handler” onpage 7-41.Plate Handler arm contactsracks when retrieving orstacking platesPlate Handler rotary offset isincorrect or vertical offset istoo lowThe Plate Handler armreleases plates awkwardlyinto the plate racksReaction plates tip or tiltwhen placed into theinstrument tray by thePlate Handler armPlate Handler fails to senseor grasp platesPlates stick to the gripperfingers of the Plate HandlerarmPlate Handler does notrestack plates in originallocationsFixed-position bar codereader not reading platebar codesPlate sensor switch notadjusted properlyGripper pads on the fingersof the Plate Handler arm areworn or dirtyGripper pads are worn ordirtyRestack when finishedoption not selectedBar code reader ismisalignedBar code reader is brokenAdjust the plate sensorswitch as explained in“Adjusting the Sensitivity ofthe Plate Sensor Switch” onpage 7-37.Change the gripper pads asexplained in “Cleaning andReplacing Gripper FingerPads” on page 7-52.Change the gripper pads asexplained in “Cleaning andReplacing Gripper FingerPads” on page 7-52.Configure the AutomationController Software torestack the plates asexplained in page 4-42.Re-align the fixed-positionbar code reader asexplained in “Aligning theFixed-Position Bar CodeReader” on page 7-49.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-17DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingTaqMan Low Density ArrayLow Density ArrayTroubleshootingTableTable 8-6Troubleshooting the TaqMan ® Low Density ArrayObservation Possible Cause Recommended ActionAfter removing the LowDensity Array from itspackaging, the fillconsumable is damaged(creased, bent, or folded).After removing the LowDensity Array from itspackaging, the aluminumfoil backing is damaged(creased, bent, or folded).After removing the LowDensity Array from itspackaging, dust or otherparticulates settle on thereaction wells (optical sideof the Low Density Array).After removing the LowDensity Array from itspackaging, watercondenses on the reactionwells (optical side of theLow Density Array).N/AN/AN/AThe Low Density Array maynot have come to roomtemperature before beingremoved from itspackaging.If the fill consumable isdamaged, then the PCRreaction mixture might notflow into the reaction wellsduring centrifugation.Discard the Low DensityArray.If the aluminum foil backingis damaged, then the PCRreaction mixture might notflow into the reaction wellsduring centrifugation.Discard the Low DensityArray.Remove dust or particulatesby lightly tapping or blowingon the reaction wells.Room-temperature,pressurized nitrogen or anair blower may be used.IMPORTANT! Be sure toremove all dust andparticulates. The reactionwells (optical side of theLow Density Array) must befree of dust andparticulates, (especiallyfluorescent particulates)Remove condensation bylightly blowing on thereaction wells.Room-temperature,pressurized nitrogen or anair blower may be used.IMPORTANT! Be sure toremove all watercondensation. The exteriorsurface of the reaction wells(optical side of the LowDensity Array) must be freeof water condensation.8-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

TaqMan Low Density ArrayTable 8-6Troubleshooting the TaqMan ® Low Density ArrayObservation Possible Cause Recommended ActionAfter pipetting, too littlePCR reaction mixture hasgone into the fill reservoir.After pipetting, some of thePCR reaction mixture leaksout of the vent port in thefill reservoir.The PCR reaction mixtureleaks during PCR cycling.After a mean assay resultand standard deviation ofthe replicates arecalculated, a specificreplicate appears to be anoutlier.The PCR reaction mixturewas not correctly pipettedinto the fill reservoir.The aluminum foil backingmay have been damagedduring the sealing step.N/ACare must be taken tocorrectly pipette the PCRreaction mixture (100 µL)into the fill reservoir.1. Carefully follow theprocedures provided in“Loading the TaqManLow Density Arrays” onpage 4-14.2. After centrifuging, besure to note the fluidlevels in the fill reservoiras described in step 6 onpage 4-18.Discard the Low DensityArray.Note: See “About theSealer” on page 4-14 forproper sealing instructions.Review the multicomponentanalysis display for thatreplicate. Delete the outlierand reanalyze.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-19DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Chapter 8TroubleshootingSDS Enterprise DatabaseSDS EnterpriseDatabaseTroubleshootingTableTable 8-7Troubleshooting the SDS Enterprise DatabaseObservation Possible Cause Recommended ActionCannot Connect to theDatabaseN/ATo test or reset the databaseconnection:1. Start the SDS software.2. Log into the database asa user withAdministrative privileges3. Test and reconfigure thedatabase connection asdescribed in“Reconfiguring theDatabase Connection”on page A-23.Error message:java.sql.SQLException:Io exception: End ofTNS data channelRare error whenarchiving or restoring alarge data setReattempt the archive orrestoration task.8-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm

Software ReferenceAAIn This Appendix Importing Plate Document Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2Setup Table Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4Exporting Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16Exporting Plate Document Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16Operating the SDS Software from a Command Line . . . . . . . . . . . . . . . . . . . . . A-18Using the Search Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-20Connecting SDS Software to the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-1DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceImporting Plate Document Setup Table FilesAbout the ImportFunctionThe SDS software features the ability to import setup table information (detector,detector task, marker, and sample name layouts) into a plate document from atab-delimited text file. The import feature is designed to be a timesaving device thatfacilitates the exchange of setup information between other programs and the SDSsoftware. Instead of setting up plate documents individually, a third-party programcan be used to construct setup table files, which can then be imported into platedocuments for use.To guarantee a successful incorporation of setup information from a text file to theplate document, the file must:• Be saved in a tab-delimited text format• Conform to the setup table file formats described on page A-15Creating andImporting SetupTable Data into aPlate DocumentImportation of setup table data into a plate document is accomplished in three majorsteps.Creating an Empty Setup Table FileThe first step in the procedure is to export a setup table file from a blank platedocument.Note: The blank setup table file can be created using a secondary application (suchas Microsoft Excel or a text editor) so long as it is saved in tab-delimited format andis configured according to the file structure explained on page A-15.1. Start the SDS software.2. Click (or select File > New).3. Configure the New Document dialog box with the correct assay type and plateformat for your experiment, and click .4. Click (or select File > Export).5. In the Look In field of the Export dialog box, navigate to the directory youwould like to receive the exported file.6. Select Export > Setup Table.7. Select the All Wells radio button.8. Select the SDS 2.2.1 radio button.9. Click the File name text box, and enter a name for the file.10. Click .The software exports the setup table data for the empty plate document as atab-delimited text file.11. Configure the setup table file with plate document information (detector, task,marker, and sample data) as explained on page A-3.A-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Importing Plate Document Setup Table FilesConfiguring the Setup Table File with Plate Document InformationThe second step in the procedure is to import the setup table file into a secondaryapplication, configure it with sample and detector information, and then save thecompleted setup table file in tab-delimited format.1. Start the application that you want to use to edit the setup table file.2. Import the setup table file from the previous procedure as tab-delimited text.If using a spreadsheet application to edit the setup table file, the applicationautomatically parses the tab-delimited information into the cells of aspreadsheet.3. Configure the setup table file with sample and detector information accordingto the file structure explained on page A-4.4. Save the setup table file in tab-delimited format.5. Import the completed setup table file into an empty plate document as explainedbelow.Importing the Completed Setup Table File into a Plate DocumentThe final step in the procedure is to import the completed setup table tab-delimitedfile into an empty plate document.1. If the plate document created in “Creating an Empty Setup Table File” onpage A-2 is still open in the SDS software, continue to step 3. Otherwise, createa plate document to receive the setup table data:a. Start the SDS software.b. Create or open a plate document to receive the information from the textfile.2. Click (or select File > Import).3. In the Look In field of the Import dialog box, navigate to and select thecompleted tab-delimited setup table file from step 4 in the previous procedure.4. Click .The software imports the setup table information from the text file andautomatically configures the plate document plate grid and setup table withdetector, detector task, marker, and sample data.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-3DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceSetup Table FilesAbout the SetupTable File FormatFormat OverviewConventionsThe SDS software features the ability to import setup table information into a platedocument from a tab-delimited text file. To guarantee a successful importation ofsetup table data, the imported file must follow the setup table format outlined in thissection.The information in the setup table file must be ordered according to the followingconvention:1. File Version (see page A-5)Plate Characteristics2. Plate Size (see page A-5)3. Plate ID (see page A-5)Detector Definitions4. Number of Detectors (see page A-6)5. Detectors List Header (see page A-6)6. Detectors List (see page A-7)Marker Definitions7. Number of Markers (Allelic Discrimination Only) (see page A-8)8. Markers List Header (Allelic Discrimination Only) (see page A-8)9. Markers List (Allelic Discrimination Only) (see page A-9)Well-Detector Information10. Well-Detector List Header (see page A-10)11. Well-Detector Definition List (see page A-10)Well-Marker Information12. Well-Marker List Header (Allelic Discrimination Only) (see page A-12)13. Well-Marker Definition List (Allelic Discrimination Only) (see page A-12)The following conventions are used in the rest of this section:• courier – Text appearing in bolded courier font must be applied to a setuptable file exactly as appears in this document.• italic – Text appearing in italic courier font must be substituted with customvalues when applied to a setup table file.• [ required text ] – Text appearing between brackets is requiredinformation in setup table files. All information inside the brackets must bepresent in the setup table file for the SDS software to import it.• { optional text } – Text appearing between braces is optional in setuptable files.• – The tab character (the equivalent of pressing the Tab key)• – The carriage-return character (the equivalent of pressing the Enter key)IMPORTANT! To guarantee a successful importation of the setup table file into aplate document, the file must contain all the elements described in the followingsection and in the order that they appear in this document.A-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table Files1. File VersionDescriptionThis line defines the version of SDS Assay Plate File format used to generate the document.Note: Assay Plate Setup Files of version 4 and higher cannot be imported directlyinto SDS 2.0 or SDS 2.1.Format [ *** SDS Setup File Version version number ]Example *** SDS Setup File Version 3Plate Characteristics2. Plate SizeDescriptionThis line defines the number of wells in the plate modeled by the file (for example,384 or 96).Format [ *** Output Plate Size number of wells ]Example *** Output Plate Size 3843. Plate IDDescriptionThis line defines the ID of the Assay Plate. Normally this is a bar code that is printedon the plate.Format [ *** Output Plate ID plate id ]Example *** Output Plate ID 384N75822034Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-5DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceDetector Definitions4. Number of DetectorsElement numbers 4 to 6 define the detectors that appear in the well descriptions thatfollow in a later section. The detector definition consists of three sections: thedeclaration of the number of detectors, the detector list header, and the detector list.DescriptionThis line defines the total number of detectors on the plate.Format [ *** Number of Detectors number of detectors ]Example *** Number of Detectors 55. Detectors List HeaderDescriptionFormatThis line contains the column headings for the Detector Definitions section of thesetup table file that make the file easier to edit using a program such as MicrosoftExcel.[ Detector Reporter Quencher Description Comments ]ExampleExample Code A-1Detectors List HeaderDetector Reporter Quencher Description CommentsA-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table Files6. Detectors ListDescriptionFormat (for asingle detector):The detector list consists of one or more lines displaying the information for eachdetector used on the plate document.• The Detectors List section must contain one line (or definition) for eachdetector present on the plate.• The number of lines in the Detectors List section must be equal to the numberdefined in the Number of Detectors section (see number 4 above).• Leave blank the Quencher Dye entry for detectors created for the SYBR Green IDye or probes labeled with a non-fluorescent quencher.[ detector name reporter dye quencher dye description comments ]Examples:Example Code A-2Allelic discrimination setup table file*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID 384N75822034*** Number of Detectors 2Detector Reporter Quencher Description CommentsCYP 2C9*2.1 FAM PDAR CYP 2C9*2 Al1 Example ProbeCYP 2C9*2.2 VIC PDAR CYP 2C9*2 Al2 Example Probe...Example Code A-3Absolute or relative quantification setup table file*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96JG729FF903*** Number of Detectors 3Detector Reporter Quencher Description CommentsGAPDH VIC GAPDH Probe Example ProbeSYBR Green SYBR SYBR Green I Example ProbeRNase P FAM TAMRA RNase P Probe Example Probe...Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-7DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceMarker DefinitionsElement numbers 7 to 9 define the markers that appear in the well descriptions thatfollow in a later section. The marker definition consists of three sections: thedeclaration of the number of markers, the marker list header, and the marker list.7. Number of Markers (Allelic Discrimination Only)IMPORTANT! Number of Markers information is required only in setup table filescreated for allelic discrimination runs. This section does not appear in setup tablefiles created for absolute or relative quantification runs.DescriptionThis line defines the total number of markers on the plate.Format [ *** Number of Markers number of markers ]Example *** Number of Markers 28. Markers List Header (Allelic Discrimination Only)IMPORTANT! Marker List Header information is required only in setup table filescreated for allelic discrimination runs. This section does not appear in setup tablefiles created for absolute or relative quantification runs.DescriptionFormatThis line contains the column headings for the Marker Definitions section of thesetup table file that make the file easier to edit using a program such as MicrosoftExcel.[ Marker AlleleX AlleleY Description Comments ]ExampleExample Code A-4Marker AlleleX AlleleY Description CommentsA-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table Files9. Markers List (Allelic Discrimination Only)IMPORTANT! Marker List information is required only in setup table files createdfor allelic discrimination runs. This section does not appear in setup table filescreated for absolute or relative quantification runs.DescriptionFormat (for asingle marker):The marker list consists of one or more lines displaying the information for eachmarker used on the plate document.• The Markers List section must contain one line (or definition) for each markerpresent on the plate.• The number of lines in the Markers List section must be equal to the numberdefined in the Number of Markers section (see number 7 above).• Leave the markers list line blank in setup files generated for relative andabsolute quantification. (Real-time plate documents do not use markers.)[ marker name alleleX alleleY description comments ]Examples:Example Code A-5Allelic discrimination setup table file*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID 384N75822034*** Number of Detectors 2Detector Reporter Quencher Description CommentsCYP 2C9*2.1 FAM PDAR CYP 2C9*2 Al1 Example ProbeCYP 2C9*2.2 VIC PDAR CYP 2C9*2 Al2 Example Probe*** Number of Markers 1Marker AlleleX AlleleY Description CommentsCYP 2C9*2 CYP 2C9*2.2 CYP 2C9*2.2 PDAR CYP 2C9*2 Example marker...Example Code A-6Absolute or relative quantification setup table file*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96JG729FF903*** Number of Detectors 3Detector Reporter Quencher Description CommentsGAPDH VIC GAPDH Probe Example ProbeSYBR Green SYBR SYBR Green I Example ProbeRNase P FAM TAMRA RNase P Probe Example ProbeApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-9DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceWell-Detector Information10. Well-Detector List HeaderElement numbers 10 and 11 define the contents of the wells on the plate in terms ofdetectors. The well-marker information consists of two sections: the Well-DetectorList Header and the Well-Detector Definition List.DescriptionThis line contains the column headings for the well-marker information section ofthe setup table file that make the file easier to edit using a program such as MicrosoftExcel.Format [ Well Sample Name Detector Task Quantity ] {... Detector Task Quantity } [ ]ExampleExample Code A-7Well Sample Name Detector Task Quantity Detector Task Quantity11. Well-Detector Definition ListDescriptionThis section defines the contents of the plate wells. The setup table file must contain adefinition for each well used on the plate. Each well definition list consists of one stringof characters terminated by a . The definition consists of three main functionaldivisions:• Well number – The first tab-delimited text block defines the number of the well onthe plate. Well numbers start at 1 for well A-1 (upper-left corner of the plate) andincreases from left to right and from top to bottom. The wells must be listed inorder (1,2,3,…).• Sample name – The second text block defines the name of the sample assigned tothe well.• Detector assignments – The remaining tab-delimited text blocks for the welldefinition define the detectors assigned to the well. Each detector is represented bythree text blocks that define the following information:– The name of the detector– The task assignment of the detector for the well (UNKN - Unknown, STND -Standard, NTC - No Template Control)– The quantity assignment of the detector for the well. (For wells containingstandards, assign the quantity for the standard sample in initial copy number. Forall other wells, assign the quantity value as 0.)To assign more than one detector to a well, repeat the detector definition text blocks foreach detector. There is no limit to the number of detectors that can appear in a well.IMPORTANT! All detectors that appear in this section must have been previouslydefined in the Detector Definitions section (elements 4–6).A-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table FilesFormat(for a single well):[ Well number SDS Sample Name Detector name Detector task Detector quantity ] { Detector name Detector task Detector quantity … Detector name Detector task Detector quantity } [ ]ExampleExample Code A-8Allelic discrimination setup table files*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID 384N75822034*** Number of Detectors 2Detector Reporter Quencher Description CommentsCYP 2C9*2.1 FAM PDAR CYP 2C9*2 Al1 Example ProbeCYP 2C9*2.2 VIC PDAR CYP 2C9*2 Al2 Example Probe*** Number of Markers 1Marker AlleleX AlleleY Description CommentsCYP 2C9*2 CYP 2C9*2.2 CYP 2C9*2.2 PDAR CYP 2C9*2 Example markerWell Sample Name Detector Task Quantity Detector Task Quantity1 Sample 12 Sample 23 ...384 Sample 48Example Code A-9Absolute quantification setup table files*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96JG729FF903*** Number of Detectors 1Detector Reporter Quencher Description CommentsRNase P FAM TAMRA RNase P Probe Example ProbeWell Sample Name Detector Task Quantity1 Sample 1 GAPDH UNKN 02 Sample 2 GAPDH STND 200003 ...96 Sample 30 GAPDH UNKN 0Example Code A-10Relative quantification setup table files*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96FRE505SDA2*** Number of Detectors 2Detector Reporter Quencher Description CommentsGAPDH VIC GAPDH Probe Example ProbeTNF-α FAM TNF-α Probe Example ProbeWell Sample Name Detector Task Quantity Detector Task Quantity1 Calibrator GAPDH ENDO 0 TNF-α UNKN 02 Sample 1 GAPDH ENDO 0 TNF-α UNKN 03 ...96 Sample 12 GAPDH ENDO 0 TNF-α UNKN 0Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-11DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceWell-Marker InformationElement numbers 12 and 13 define the contents of the wells on the plate in terms ofmarkers. The well-marker information consists of two sections: the Well-Marker ListHeader and the Well-Marker Definition List.12. Well-Marker List Header (Allelic Discrimination Only)IMPORTANT! Well-Marker List Header information is required only in setup tablefiles created for allelic discrimination runs. This section does not appear in setuptable files created for absolute or relative quantification runs.DescriptionThis line contains the column headings for the well-marker information section of thesetup table file that make the file easier to edit using a program such as Microsoft Excel.Format [ Well Sample Name Detector Task Quantity ] {... Detector Task Quantity } [ ]ExampleExample Code A-11Well Sample Name Marker Task Marker Task13. Well-Marker Definition List (Allelic Discrimination Only)IMPORTANT! Well-Marker Definition List information is required only in setuptable files created for allelic discrimination runs. This section does not appear insetup table files created for absolute or relative quantification runs.DescriptionThis section defines the contents of the plate wells. The setup table file must contain adefinition for each well used on the plate. Each well definition list consists of one stringof characters terminated by a . The definition consists of three main divisions:• Well number – The first tab-delimited text block defines the number of the well onthe plate. Well numbers start at 1 for well A-1 (upper-left corner of the plate) andincreases from left to right and from top to bottom. The wells must be listed inorder (1,2,3,…).• Sample name – The second text block defines the name of the sample assigned tothe well.• Marker assignments – The remaining tab-delimited text blocks for the welldefinition define the marker assigned to the well. Each marker is represented bytwo text blocks that define the following information:– The name of the marker– The task assignment of the marker for the well (UNKN - Unknown, NTC - NoTemplate Control)To assign more than one marker to a well, repeat the marker definition text blocks foreach detector. There is no limit to the number of markers that can appear in a well.IMPORTANT! All markers that appear in this section must have been previouslydefined in the Marker Definitions section (elements 7–9).A-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table FilesFormat(for a single well):[ Well number SDS Sample Name Marker name Marker task ]{ Marker name Marker task … } [ ]ExampleExample Code A-12Allelic discrimination setup table files*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID 384N75822034*** Number of Detectors 2Detector Reporter Quencher Description CommentsCYP 2C9*2.1 FAM PDAR CYP 2C9*2 Al1 Example ProbeCYP 2C9*2.2 VIC PDAR CYP 2C9*2 Al2 Example Probe*** Number of Markers 1Marker AlleleX AlleleY Description CommentsCYP 2C9*2 CYP 2C9*2.2 CYP 2C9*2.2 PDAR CYP 2C9*2 Example markerWell Sample Name Detector Task Quantity1 Sample 12 Sample 23 ...384 Sample 48Well Sample Name Marker Task Marker Task1 Sample 1 CYP 2C9*2 UNKN2 Sample 2 CYP 2C9*2 UNKN3 ...384 Sample 48 CYP 2C9*2 UNKNExample Code A-13Absolute quantification setup table files*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96JG729FF903*** Number of Detectors 1Detector Reporter Quencher Description CommentsRNase P FAM TAMRA RNase P Probe Example ProbeWell Sample Name Detector Task Quantity Detector Task Quantity1 Sample 1 GAPDH UNKN 02 Sample 2 GAPDH STND 200003 ...96 Sample 30 GAPDH UNKN 0Example Code A-14Relative quantification setup table files*** SDS Setup File Version 3*** Output Plate Size 96*** Output Plate ID 96FRE505SDA2*** Number of Detectors 2Detector Reporter Quencher Description CommentsGAPDH VIC GAPDH Probe Example ProbeTNF-α FAM TNF-α Probe Example ProbeWell Sample Name Detector Task Quantity Detector Task Quantity1 Calibrator GAPDH ENDO 0 TNF-α UNKN 02 Sample 1 GAPDH ENDO 0 TNF-α UNKN 03 ...96 Sample 12 GAPDH ENDO 0 TNF-α UNKN 0Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-13DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceExample Setup Table FilesExample Code A-15Relative quantification setup table document*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID 384JQZ55A4*** Number of Detectors 25Detector Reporter Quencher Description Comments18S-VIC-mgb VICBcl-2 FAM Human Bcl-2Bcl-xL FAM Human Bcl-xLGAPDH FAM Human GAPDHGBP2 FAM guanlylate binding protein 2GOS2 FAM Human GOS2 protein geneIL-15 FAM Human IL-15IL-17 FAM Human IL-17IL-18 FAM Human IL-18IL-1a FAM Human IL-1aIL-1b FAM Human IL-1 betaIL12p40 FAM Human IL-12p40INF-a2 FAM Interferon-alpha2IP-10 FAM Human IP-10ISG56 FAM Interferon-induced protein 56Nfkb-I FAM Nuclear factor of kappa light...PKCi FAM Protein kinase C, iotaRAP1A FAM RAP1A, member of RAS oncogene...RhoPK2 FAM Homo sapiens Rho-associated...TNF-a FAM Human TNF-alphac-fos FAM Human c-fosc-jun FAM Human c-junc-myc FAM Human c-myccot FAM Cancer Osaka thyroid oncogenep53 FAM Human p53Well Sample Name Detector Task Quantity1 A1 18S-VIC-mgb ENDO 0.02 Liver 18S-VIC-mgb ENDO 0.0 GOS2 TARG 0.03 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.04 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.05 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.06 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.07 Liver 18S-VIC-mgb ENDO 0.0 GOS2 TARG 0.08 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.09 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.010 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.011 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.012 Liver 18S-VIC-mgb ENDO 0.0 GOS2 TARG 0.013 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.014 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.015 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.016 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.0...381 Liver 18S-VIC-mgb ENDO 0.0 GOS2 TARG 0.0382 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.0383 Liver 18S-VIC-mgb ENDO 0.0 cot TARG 0.0384 Liver 18S-VIC-mgb ENDO 0.0 ISG56 TARG 0.0Information for wells 17 through 380 has been removed from this example for brevity.A-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Setup Table FilesExample Code A-16Allelic discrimination setup table document*** SDS Setup File Version 3*** Output Plate Size 384*** Output Plate ID NU03000081*** Number of Detectors 8Detector Reporter Quencher Description CommentsAL2-1FAMAL1-1VICAL2-2FAMAL1-2VICAL2-3FAMAL1-3VICAL2-4FAMAL1-4VIC*** Number of Markers 4Marker AlleleX AlleleY Description CommentsSNP1 AL2-1 AL1-1SNP4 AL2-4 AL1-4SNP3 AL2-3 AL1-3SNP2 AL2-2 AL1-2Well Sample Name Detector Task Quantity1 A12 A23 A34 A45 A56 A67 A78 A89 A910 A1011 A1112 A1213 A13...382 P22383 P23384 P24Well Sample Name Marker Task1 A1 SNP1 UNKN2 A2 SNP1 UNKN3 A3 SNP1 UNKN4 A4 SNP1 UNKN5 A5 SNP1 UNKN6 A6 SNP1 UNKN7 A7 SNP1 UNKN8 A8 SNP1 UNKN9 A9 SNP1 UNKN10 A10 SNP1 UNKN11 A11 SNP1 UNKN12 A12 SNP1 UNKN13 A13 SNP1 UNKN...382 P22 SNP4 UNKN383 P23 SNP4 UNKN384 P24 SNP4 UNKNInformation for wells 11 through 381 has been removed from this example for brevity.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-15DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceExporting GraphicsExporting a Plotas a JPEGGraphic FileThe SDS software can export most panes and plots of the plate document as JPEG(Joint Photographic Experts Group) graphic files. The JPEG file format iscompatible with most word processing and spreadsheet applications and can beincorporated directly into HTML documents for viewing by most web browsersoftware.To export an element of a plate document as a graphic:1. Select the plot or grid you want to export.2. Choose from the following:– If exporting a plot, adjust its dimensions (length and width) as you want themto appear in the exported graphic file. The exported graphic file retains thedimensions of the original screen element.– If exporting the plate grid, do not adjust the size of the wells. The softwarecaptures the whole grid regardless of the size of the view.3. Right-click the plot or grid, and select Save Plot/Grid to Image File from thecontextual menu.Note: If a pane cannot be exported as a graphic, the contextual menu will notcontain the Save Plot/Grid to Image File option.4. In the Save As dialog box, navigate to the directory you want to receive theexported graphic file.5. In the File name field, and enter a name for the new file.6. Click .The software saves the plot or grid as a JPEG graphic in the designateddirectory.Exporting Plate Document DataExporting Datafrom a PlateDocumentThe SDS software can export raw or analyzed data in tab-delimited (*.txt) format forall or a select group of wells on a plate document. The exported files are compatiblewith most spreadsheet applications.To export an element of a plate document:1. Click the plate document to select it.IMPORTANT! The plate document must be the top-most object in the workspace.2. Click (or select File > Export).3. Navigate to the directory you would like to receive the exported file(s).A-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Exporting Plate Document Data4. In the Export drop-down list, select the type of data you would like to export.• Background Spectra – The exported file contains the fluorescence readingsfor each well from the background component used to analyze the run• Clipped – The exported file contains the average R n and ∆R n of the lastthree data points collected during the extension phase of each cycle repetitionfor each well• Dissociation Curve – The exported file contains:Temperature Data – For each well in use on the plate, the file displays thecalculated temperature of the wells during each data collection reading of thetemperature ramp.Raw Data – For each well in use on the plate, the file displays the R n of thewell during each data collection reading of the temperature ramp.Derivative Data – For each well in use on the plate, the file displays the firstderivative data during each data collection reading of the temperature ramp.• Multicomponent – The exported file contains:– Amounts the calculated dye components in a single well throughout allstages of the PCR that were labeled with data collection icons– Pure spectra component data– Calculated inverse matrix– Singular values of the inverse matrix• Pure Spectra – The exported file contains the fluorescence readings for eachwell from the pure spectra calibration component used to analyze the run• Raw Spectra – The exported file contains the unmodified fluorescencereadings taken for each spectral bin during the course of the run• When exported, the software creates a directory and saves each the rawspectra data for each well in a separate *.txt file.• Results Table – The exported file contains the contents of the table pane ofan analyzed plate documentNote: The contents of the exported data vary depending on the type of platedocument.• Setup Table – The exported file contains the contents of the table pane of aplate document before analysis The contents of the exported data variesdepending on the type of plate document used to produce it. See page A-4 fora detailed description of the Setup Table file.5. To export data from:• All wells of the plate document – Select the All Wells radio button.• Selected wells of the plate grid – Select the Selected Wells radio button.6. In the files of type drop-down list, select the appropriate format for the exporteddata.7. Click the File name text box, and enter a name for the exported file.8. Click .The software saves the exported data to the designated location.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-17DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceOperating the SDS Software from a Command LineOverviewRunning theSoftware from aCommand LineExample Code A-17The SDS software allows you to use batch files to analyze and export results from thecommand line. Use of the command line interface is intended for advanced users(such as systems administrators, bioinformaticians, and laboratory networkadministrators) who choose to drive the application using a scripting language.Note: If you are unfamiliar with Microsoft DOS, Applied Biosystems recommendsrunning the application from the user interface.1. In the desktop, double-click My Computer > AppliedBiosystems > SDS2.2.1.2. Create a copy of the SDS 2,bat file and rename it (such as SDS_RESULTS.bat).3. Using the Notepad application, edit the contents of the batch file as follows.SDS2.bat Batch FileThe text of the batch file appears as follows:set classpath=./service/config;./service/Lib/audit.jar;./service/Lib/rtc.jar;./service/Lib/ab-reqres.jar;./service/Lib/translation.jar;./service/Lib/apv.jar;./service/lib/coreutil.jar;./service/JMS/lib/fmprtl.zip;./service/Lib/coregui.jar;./service/lib/EmlException.jar;./service/lib/emlhandler.jar;./service/lib/esig.jar;./service/lib/esig/exception.jar;./service/lib/Coreutil.jar;set path=./lib/algorithm/bin/win32;./lib;%path%jre\bin\java -Dab.home=C:\AppliedBiosystems\SDS2.2\service -Dcom.apldbio.sds.global.approot=.-Dcom.apldbio.sds.global.config= .\config\config.properties -Xms110m -Xmx300m-Dsource=batchfile -Ddefport=8080 -classpath %classpath%;lib\SDS2.jar;lib\common.jar;lib\sdsdb.jar;lib\ojdbc14.jar;lib\accesscontrol.jar;lib\sdsdb.jar;lib\activation.jar;lib\mail.jar;lib\accesscontrol.jar;lib\Coreutil.jar;lib\EmlException.jarcom.apldbio.sds.shell.SDSMainIn the last line, replacecom.apldbio.sds.shell.SDSMainwith one of the following, to generate:• Multicomponent data for an SDS plate document file, enter:com.apldbio.sds.MulticompAnalyzerMain %*• Analyzed data for an SDS plate document file, enter:com.apldbio.sds.SDSResultsAnalyzerMain %*• Multicomponent and analyzed data for a plate document file, enter:com.apldbio.sds.MulticompAndResultsAnalyzerMain %*4. Save and close the batch file.5. Invoke the file at the command line by entering:SDS_RESULTS.bat sdsfile.sds sdsresultsfile.txtwhere:• SDS_RESULTS.bat – Is the name of the batch file you created.• sdsfile.sds – Is the SDS plate document file that you have targeted foranalysis.• sdsresultsfile.txt – Is the name of the exported results file.A-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Operating the SDS Software from a Command LineEnhancing thePerformance ofthe SDS SoftwareIt is possible to enhance the performance (processing speed) of the SDS software byincreasing the memory partition allocated for the software. To increase the memorymade available to the SDS software, insert the parameters shown in the examplebelow into any command issued to the software.jre\bin\java -Dab.home=C:\AppliedBiosystems\SDS2.2\service-Dcom.apldbio.sds.global.approot=. -Dcom.apldbio.sds.global.config=.\config\config.properties -Xms80m -Xmx90m -Dsource=batchfile-Ddefport=8080…where:• -Xms80m – Is the minimum amount of memory that the operating system willallocate for the SDS software.• -Xmx90m – Is the maximum amount of memory that the operating system willallocate for the SDS software.Applied Biosystems recommends the following settings:• End-point runs (allelic discrimination): -Xms20m -Xmx60m• Real-time runs (absolute or relative quantifiation): -Xms80m -Xmx90mApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-19DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceUsing the Search ToolProgrammingFiltersSearchStatementsSearch StatementSyntaxThe Search tool helps to organize the large set data in the Add files dialog box. TheSearch tool allows the software to display a subset of the total number of platedocuments based on a set of predefined criteria.The Search tool operates on a logic similar to a simple computer programminglanguage. Each filter consists of a single logical statement that instructs the softwareto include or exclude plate documents based on a specific parameter or quality. Thestatements can be concatenated to screen the displayed data based on multiplecriteria.The Search tool consists of a table in which each row represents a single statementthat tests the associated data set for a condition. Every time a row (or statement) isadded to the table, the software evaluates each plate document in the selecteddirectory for the condition defined by the statement. If a plate document meets thecriteria defined by the test (the test evaluates true), then the software displays it in theSearch Results list. If a plate document does not meet the criteria (the test evaluatesfalse), the software hides the document from view and excludes the data from theanalysis. As explained later in this section, the Search tool can evaluate multipleexpressions (many rows) to refine the data set.Figure A-1 shows a filter created in the Add Files dialog box that illustrates the basicstructure of all statements.Logic OperatorA logical operator(IF, AND, or OR)Column CategoryDescribes the categoryfor the evaluationConditional OperatorDescribes the nature ofthe evaluationValue CriteriaContains the conditionfor the evaluationFigure A-1Add Files dialog boxA-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Using the Search ToolLogical Operators(IF, AND, OR)Every statement begins with one of three logical operators: IF, OR, or AND. Thelogical operator determines the relationship between the statements in the Add Filesdialog box.Logical IF StatementsBy default, the first statement in the table is always an IF statement. If a platedocument meets the criteria defined by the statement, it is displayed within the AddFiles dialog box.Logical AND StatementsA logical AND statement evaluates the data set for two expressions: the expressiondefined by the AND statement, and the expression defined by the statement in theprevious table row. When an AND statement is used in a filter table, the softwaredisplays a plate document if it evaluates true for both expressions.Logical OR StatementsSimilar to the logical AND statement, a logical OR statement also evaluates the data setfor two expressions (both the previous and current table rows). When an OR statementis used in a Filter table, the software displays a plate document if it evaluates true foreither expression (or both).Multiple AND and OR StatementsIf dealing with a very large data set, you may want to further refine the number ofplate documents retrieved in a search by creating multiple filter statements. Whencreating multiple statements, remember to create and apply them one at a time toensure that they function as expected. Also, remember that the software applies eachfilter in the order listed in the Add Files dialog box.ColumnCategoriesConditionOperatorsValue 1 and 2CriteriaEach statement evaluates plate documents based on the values of specific platedocument attributes.The Condition operator establishes the relationship between the Column categoryand the Value 1 and Value 2 criteria by defining the action of the evaluation. Thefollowing list summarizes the possible conditional operators and their correspondingactions.• > : Greater than Value 1• >= : Greater than or equal to Value 1• < : Less than Value 1• : Does not equal Value 1• between : Is greater than Value 1 but less than Value 2• contains : Contains the alphanumeric string found in Value 1The Value 1 and Value 2 criteria define the condition(s) for the evaluation.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-21DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware ReferenceConnecting SDS Software to the DatabaseRequiredInformationDisabling/Enablingthe DatabaseFunctions in theSDS SoftwareIMPORTANT! Only users belonging to the Administrator or Service Engineer usergroup can modify the database parameters for an SDS client application.You need the following information to connect a SDS client application to the database:• Host Name – The Host Name setting or the IP address for the SDS EnterpriseDatabase. Check with your database administrator for the correct identificationnotation for the server running the database on your network.• Port Number – The Port Number default setting is 1521. (You may need tocheck with your database administrator if the port setting has been modified.)• Service Name – The Service Name setting is configured during the installationof the SDS Enterprise Database. The default is SDS22.The SDS software allows users of the Administrator User Group to disable thedatabase functions of the software. When disabled, the software hides the toolbarbuttons and menu commands for opening and saving data to the database.Note: The enable/disable feature can be used during periods when the networkconnection is unavailable. By disabling the database connection, the SDS softwaredoes not require users to log in before using the 7900HT instrument. After thedatabase becomes available again, the data collected in the interim can be uploadedto the SDS Enterprise Database using the SDS Document Loader. For moreinformation, see the SDS Enterprise Database for the Applied Biosystems 7900HTFast Real-Time PCR System Administrators Guide (P/N 4351669).To disable or enable the database features of the SDS software:IMPORTANT! You must belong to the Administrators User Group to modify thedatabase options.1. Start the SDS software (see page 2-7 for more information).2. In the Login dialog box, enter a user name and password of a user account thatbelongs to the Administrator user group (see the SDS Enterprise Database forthe Applied Biosystems 7900HT Fast Real-Time PCR System AdministratorsGuide for more information on user accounts).3. Select Tools > Options.4. In the Options dialog box, select the Database tab.5. Select or deselect the Enable Enterprise Options check box to activate ordeactivate the database features.6. Click .7. When prompted, enter your user name and password, then click .A-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Connecting SDS Software to the DatabaseReconfiguring theDatabaseConnectionIMPORTANT! You must have Administrative privileges to reconfigure the SDSEnterprise Database connection (see the SDS Enterprise Database for the AppliedBiosystems 7900HT Fast Real-Time PCR System Administrators Guide for moreinformation on user accounts).The connection to the SDS Enterprise Database can be reconfigured from theOptions dialog box of the SDS software as explained below.1. Start the SDS software (see page 2-7 for more information).2. In the Login dialog box, enter a user name and password of a user account thatbelongs to the Administrator user group.3. Select Tools > Options.4. In the Options dialog box, select the Database tab.5. Configure the Current Selected Connection settings:Host Name fieldEnter the host name orIP address of the databasePort Number fieldEnter the port number of thedatabase connection(default is 1521)Service Name fieldEnter the name of the database(default is SDS22)6. Test the new connection:a. Click to test the new connection.The software briefly checks that a database is at the given IP address. If theconnection is successful, the software displays the Login dialog box.b. In the Test Login dialog box, enter your User Name and Password, thenclick .• If the SDS software is unable to successfully connect to the database, thesoftware displays the error message shown below.7. In the Options dialog box, click .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-23DRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Appendix ASoftware Reference8. Log into the database by entering your user name and password, thenclick .A-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_SWReference.fm

Designing TaqMan Reagent-BasedAssaysBBIn This AppendixAssay Development Guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2Design Tips for Allelic Discrimination Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5Design Tips for Quantitative PCR Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-6Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-1DRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Appendix BDesigning TaqMan Reagent-Based AssaysAssay Development GuidelinesTaqMan Reagent-Based AssayDevelopmentProgramIdentify TargetSequence(s)Design Probesand Primers1. Identify target sequence(s) (see page B-2).2. Design the TaqMan ® probes and the forward and reverse primers (see page B-2).3. Order reagents.4. Quantitate the concentrations of the probes and primers (see page B-3).5. Prepare the master mix (see page B-3).6. Optimize the primer concentrations (see page B-3).7. Run the assay (see page B-4).A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotidesequence of interest. For optimal results, the target template should meet thefollowing requirements:• The target nucleotide sequence must contain binding sites for both primers(forward and reverse) and the fluorogenic probe.• Short amplicons work best. Amplicons ranging from 50–150 bp typically yieldthe most consistent results.• If designing assays for quantitative PCR, see “Design Tips for Quantitative PCRAssays” on page B-6 for additional recommendations.The following sections contain general guidelines for designing primers and probes.For specific design tips, refer to the appropriate section: for Allelic Discriminationsee page B-5 and for Quantitative PCR see page B-6.Design Probe(s) for the AssayAdhere to the following guidelines when designing TaqMan probes:• Keep the G-C content in the range of 30–80%.• Avoid runs of an identical nucleotide (especially guanine, where runs of four ormore Gs should be avoided).• No G on 5´ end.• Keep the melting temperature (T m ) in the range of 68-70 °C for quantitative PCRand 65-67 °C for allelic discrimination (using the Primer Express software).• Select the strand that gives the probe with more Cs than Gs.• For allelic discrimination (see page B-5):– Adjust probe length so that both probes have the same T m .– Position the polymorphism site approximately in the center of each probe.• For absolute or relative quantification (see page B-6)B-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Assay Development Guidelines• For multiplex PCR applications (involving multiple probes), design the probeswith different fluorescent reporter dyes (see Table B-1).Table B-1Reporter Dyes for Multiplex PCR ApplicationsFirst ProbeSecond ProbeReporter Dye *FAM VIC ®*The use of the FAM and VIC reporter dyes for multiplex applications provides the greatestdegree of spectral separation.Design Primers for the AssayAdhere to the following guidelines when designing primers for 5´-nuclease assays:• Keep the G-C content in the range of 30–80%.• Avoid runs of an identical nucleotide (especially guanine, where runs of four ormore bases should be avoided).• Keep the T m in the range of 58-60 °C (using the Primer Express software).• Limit the G and/or C bases on the 3´ end. The five nucleotides at the 3´ endshould have no more than two G and/or C bases.• Place the forward and reverse primers as close as possible to the probe withoutoverlapping the it.• Use an annealing temperature of 58-60 °C for quantitative PCR, and 58-60 °C forallelic discrimination (except for TaqMan ® PDARs for Allelic Discrimination).Quantitate theProbes andPrimersPrepareMaster MixOptimizePrimer/ProbeConcentrationsUse a spectrophotometric method to determine the concentrations of the probes andprimers received. See the TaqMan ® Universal PCR Master Mix Protocol(PN 4304449) or the TaqMan ® Fast Universal PCR Master Mix (2✕) Protocol(PN 4351891) for specific information about primer and probe quantification.Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) or theTaqMan ® Fast Universal PCR Master Mix (2✕) Protocol (PN 4351891) for specificinformation about preparing the master mix for use.IMPORTANT! PCR master mix used with the 7900HT instrument must contain apassive reference dye. The SDS software uses the signal from the passive referenceto normalize the reporter fluorescence making well-to-well comparisons possible.All Applied Biosystems master mix products contain an optimal concentration of theROX passive reference dye.Note: Applied Biosystems protocols are available on the Applied BiosystemsCompany Web Site. See “How to Obtain Services and Support” on page xii for moreinformation.Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) forspecific information about optimizing primer/probe concentrations.Note: Applied Biosystems protocols are available on the Applied BiosystemsCompany Web Site. See “How to Obtain Services and Support” on page xii for moreinformation.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-3DRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Appendix BDesigning TaqMan Reagent-Based AssaysRun Your CustomAssayNote: If conducting a quantitative PCR experiment, consider the use of replicateassays to enhance the precision of you data.B-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Design Tips for Allelic Discrimination AssaysDesign Tips for Allelic Discrimination AssaysDiscrimination byMultiple ProbesTaqMan ProbeDesignGuidelinesBy using different reporter dyes, cleavage of multiple probes can be detected in asingle PCR. One application of this multi-probe capability is to use allele-specificprobes to distinguish genetic polymorphisms (Bloch, 1991, Lee et al., 1993). Probesthat differ by as little as a single nucleotide will exhibit allele-specific cleavage. Thisis true even for probes with a reporter on the 5' end and the non-fluorescent quencheron the 3´ end (Bloch, 1991).IMPORTANT! When designing probes, it is important to consider probes from bothstrands.Follow the guidelines in the table below for designing TaqMan MGB probes:Table B-2Priority1Guidelines for Designing TaqMan MGB Probes for Allelic DiscriminationGuidelineAvoid probes with a guanine residue at the 5´ end of the probe.A guanine residue adjacent to the reporter dye will quench the reporterfluorescence, even after cleavage.2 Select probes with a Primer Express software–estimated T m of 65–67 °C.34Make the TaqMan MGB probes as short as possible, but no fewer than13 nucleotides in length.Avoid runs of an identical nucleotide.This is especially true for guanine, where runs of four or more should beavoided.Position the polymorphic site in the central third of the probe.Note: The polymorphic site can be shifted toward the 3´ end to meet theabove guidelines, however, the site must be located more than twonucleotides upstream from the 3´ terminus.The following figure illustrates the placement of a polymorphism in an exampleprobe (N = Nucleotide).5First, try to position the polymorphicsite in the central third of the probe.Do not placeit here.5´ 3´N N N N N N N N N N N N N N N N N N N N NPolymorphismIf necessary, place thepolymorphism here.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-5DRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Appendix BDesigning TaqMan Reagent-Based AssaysDesign Tips for Quantitative PCR AssaysSelecting anAmplicon Site forGene ExpressionAssaysSelecting andPreparingStandards forAbsoluteQuantificationSelecting a good amplicon site ensures amplification of the target mRNA withoutco-amplifying the genomic sequence, pseudogenes, and related genes.Applied Biosystems recommends the following guidelines when selecting anamplicon site for quantification assays:• Primers and probes must be designed following the “Assay DevelopmentGuidelines” on page B-2.• The amplicon should span one or more introns to avoid amplification of thetarget gene in genomic DNA.• The primer pair has to be specific to the target gene and does not amplifypseudogenes or other related genes.• Test amplicons and select those that have the highest signal-to-noise ratio (suchas those yielding low C T s with cDNA and no amplification with no templatecontrol or genomic DNA).• If no good sequence is found, it may be necessary to examine the sequence andredesign the amplicon or simply screen for more sites.If the gene you are studying does not have introns, then you cannot design anamplicon that will amplify the mRNA sequence without amplifying the genomicsequence. In this case, it may be necessary to run RT minus controls.To ensure accurate results, the standards used for absolute quantification must becarefully engineered, validated, and quantified before use. Consider the followingcritical points for the proper use of absolute standard curves:• The DNA or RNA used must be a single, pure species. For example, plasmidDNA prepared from E. coli often is contaminated with RNA, which increasesthe A 260 measurement and inflates the copy number determined for the plasmid.• In general, DNA cannot be used as a standard for absolute quantification ofRNA because there is no control for the efficiency of the reverse transcriptionstep.• Absolute quantities of the standard must be known by some independent means.Plasmid DNA or in vitro transcribed RNA are commonly used to prepareabsolute standards. Concentration is measured by A 260 and converted to thenumber of copies using the molecular weight of the DNA or RNA.• Consider the stability of the diluted standards, especially for RNA. Dividediluted standards into small aliquots, store at -80 °C, and thaw only once beforeuse. An example of the effort required to generate trustworthy standards isprovided by Collins et al. (Anal. Biochem. 226:120-129, 1995), who reportedon the steps they used in developing an absolute RNA standard for viralquantification.• Pipetting must be accurate because the standards must be diluted over severalorders of magnitude. Plasmid DNA or in vitro transcribed RNA must beconcentrated in order to measure an accurate A 260 value. The concentrated DNAor RNA must then be diluted 10 6 –10 12 -fold to be at a concentration similar tothe target in biological samples.B-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm

Kits, Reagents, and ConsumablesCCIn This AppendixInterchangeable Sample Block Modules and Accessories . . . . . . . . . . . . . . . . . . .C-2Consumables and Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-3Instrument Maintenance and Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-5TaqMan Pre-Developed Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-6Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-6Note: Part numbers listed in this appendix are for customers inside the UnitedStates. Contact your Regional Sales Office for local Part numbers and prices. See“How to Obtain Services and Support” on page xii.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide C-1DRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Appendix CKits, Reagents, and ConsumablesInterchangeable Sample Block Modules and AccessoriesThe 7900HT instrument features a Peltier-based, interchangeable sample block modulebased on the technology established in the GeneAmp ® PCR System 9700 thermal cycler.The use of an interchangeable sample block module:• Reduces instrument downtime by allowing immediate replacement of the block.• Permits easy access to the sample block for troubleshooting and maintenance(see page 7-14).• Supports multiple consumable formats.• Provides several different modes of operation (including Max mode andprogrammable temperature ramps).Table C-1Sample Block Modules and AccessoriesPart No. Description Quantity4331406 7900HT System Standard 384-Well Block Upgrade KitIncludes a Standard 384-Well Block, a 384-well plate adapter,and a Sequence Detection Systems 384-Well SpectralCalibration Kit (PN 4323977)4331405 7900HT System Standard 96-Well Block Upgrade KitIncludes a Standard 96-Well Block, a 96-well plate adapter,and an ABI PRISM 7900HT Sequence Detection Systems96-Well Spectral Calibration Kit (PN 4328639)4351402 7900HT System Fast 96-Well Block Upgrade KitIncludes a Fast 96-Well Block, a Fast 96-Well Plate Adapter, a7900HT System Fast 96-Well Spectral Calibration Kit(PN 4351653), a TaqMan ® RNase P Fast 96-Well InstrumentVerification Plate (PN 4351979), and a TaqMan ® FastReagents Starter Kit (PN 4352407)1kit1kit1kit4329012 7900HT System TaqMan ® Low Density Array Upgrade 1 kitC-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Consumables and DisposablesConsumables and DisposablesThe Applied Biosystems 7900HT Fast Real-Time PCR System can run:• ABI PRISM 384-Well Optical Reaction Plates sealed with optical adhesivecovers• ABI PRISM 96-Well Optical Reaction Plates sealed with optical adhesivecovers or ABI PRISM Optical Caps (flat cap strips only)• Optical 96-Well Fast Thermal Cycling Plates sealed with optical adhesivecovers• TaqMan ® Low Density ArraysThe optical plates recommended above are designed specifically forfluorescence-based PCR chemistries and are frosted to minimize external fluorescentcontamination. Before running prepared optical plates on the 7900HT instrument,each plate must be sealed with the optical adhesive covers recommended above.Applied Biosystems optical adhesive covers are specifically designed to permit thetransmission of light to and from the wells of the optical plate.IMPORTANT! Do not use MicroAmp ® Optical Caps or MicroAmp ® Optical Tubeswith the 7900HT instrument. The instrument is not designed to run MicroAmpconsumables which may damage its internal components if used.Table C-2Consumables and Disposables for the 7900HT InstrumentPart No. Description QuantityOptical Adhesive Covers4313663 ABI PRISM Optical Adhesive Cover Starter KitIncludes 20 ABI PRISM Optical Adhesive Covers, anApplicator, and an ABI PRISM Optical Cover CompressionPad.20 Covers4311971 ABI PRISM Optical Adhesive Covers 100 Covers4360954 Optical Adhesive Covers 25 Covers4323032 ABI PRISM Optical Caps, 8 Caps/Strip 300 Strips/Pkg2400 Caps/Pkg384-Well Optical Reaction Plates4309849 ABI PRISM 384-Well Clear Optical Reaction Plate withBarcode (code 128)4326270 ABI PRISM 384-Well Clear Optical Reaction Plate withBarcode (code 128), 10-PackIncludes 10 of PN 4309849, ABI PRISM 384-Well ClearOptical Reaction Plates with Barcode50 Plates500 PlatesApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide C-3DRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Appendix CKits, Reagents, and ConsumablesTable C-2Consumables and Disposables for the 7900HT InstrumentPart No. Description Quantity96-Well Optical Reaction Plates4306737 ABI PRISM 96-Well Optical Reaction Plate with Barcode(code 128)4326659 ABI PRISM 96-Well Optical Reaction Plate with Barcode(code 128), 25-PackIncludes 25 of PN 4306737, ABI PRISM 96-Well OpticalReaction Plates with Barcode4314320 ABI PRISM 96-Well Optical Reaction Plate with Barcode(code 128) and ABI PRISM Optical Adhesive CoversIncludes 100 ABI PRISM Optical Adhesive Covers(PN 4311971) and 5 of PN 4306737, ABI PRISM 96-WellOptical Reaction Plates with Barcode4312063 MicroAmp ® Splash Free Support Base for 96-Well ReactionPlates20 Plates500 Plates100 Plates100 Covers10 BasesOptical 96-Well Fast Plates4346906 Optical 96-Well Fast Thermal Cycling Plate with Barcode(code 128)4312063 MicroAmp ® Splash Free Support Base for 96-Well ReactionPlates20 Plates10 BasesC-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Instrument Maintenance and VerificationInstrument Maintenance and VerificationThe following sequence detection kits and reagents are used to perform routinemaintenance on and verify the function of the Applied Biosystems 7900HT FastReal-Time PCR System. For more information about the use of the kits below, seeChapter 7, “Maintaining the Instrument.”Table C-3Consumables for Instrument Maintenance and VerificationPartNumberDescriptionQuantitySequence Detection Systems Spectral Calibration Kits4328639 ABI PRISM 7900HT Sequence Detection Systems 96-WellSpectral Calibration KitIncludes three ABI PRISM 96-Well Optical Reaction Plates:one preloaded and sealed background plate, and twopreloaded and sealed Spectral Calibration plates containingeight separate dye standards (FAM , JOE , NED , ROX ,SYBR ® Green, TAMRA , TET , VIC ® ).4323977 Sequence Detection Systems 384-Well Spectral CalibrationKitIncludes two ABI PRISM 384-Well Optical Reaction Plates:one preloaded and sealed background plate and onepreloaded and sealed Spectral Calibration plate containingeight separate dye standards (FAM, JOE, NED, ROX, SYBRGreen, TAMRA, TET, VIC).4351653 7900HT System Fast 96-Well Spectral Calibration KitIncludes three Optical 96-Well Fast Thermal Cycling Plates:one preloaded and sealed background plate, and twopreloaded and sealed pure dye plates containing eightseparate dye standards (FAM , JOE , NED , ROX , SYBR ®Green, TAMRA , TET , VIC ® ).3 x 96-WellPlates2x384-WellPlates3 x 96-WellPlatesTaqMan RNase P Instrument Verification Plates4310982 TaqMan ® RNase P Instrument Verification PlateIncludes one ABI PRISM 96-Well Optical Reaction Plate. Eachwell contains preloaded reaction mix (master mix, RNase Pprimers, and FAM dye-labeled probe) and template to detectand quantitate genomic copies of the human RNase P gene.4323306 TaqMan ® RNase P 384-Well Instrument Verification PlateIncludes one ABI PRISM 384-Well Optical Reaction Plate.Each well contains preloaded reaction mix (master mix,RNase P primers, and FAM dye-labeled probe) and templateto detect and quantitate genomic copies of the humanRNase P gene.1 x 96-WellPlate1 x384-WellPlateApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide C-5DRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Appendix CKits, Reagents, and ConsumablesTable C-3Consumables for Instrument Maintenance and VerificationPartNumberDescriptionQuantity4351979 TaqMan ® RNase P Fast 96-Well Instrument Verification PlateIncludes one Optical 96-Well Fast Thermal Cycling Plate.Each well contains preloaded reaction mix (master mix,RNase P primers, and FAM dye-labeled probe) and templateto detect and quantitate genomic copies of the humanRNase P gene.1 x 96-WellPlateTaqMan ® Low Density 7900HT Installation Array (TGF-β card)TaqMan Pre-Developed Assays and ReagentsFor the latest information on TaqMan PDARs covering gene expressionquantification and allelic discrimination, visit the TaqMan PDAR list on theApplied Biosystems web site at:• www.appliedbiosystems.com/pdarlistCustom Oligonucleotide SynthesisTo order custom oligonucleotides:• Visit the Applied Biosystems Online Store(http://store.appliedbiosystems.com), or• Email Applied Biosystems with your order(OligosUS@appliedbiosystems.com)Table C-4Custom OligonucleotidesPartNumberDescriptionTaqMan ® MGB Probes (5’-Fluorescent label: 6-FAM, VIC or TET)4316034 5,000-6,000 pmols4316033 15,000-25,000 pmols4316032 50,000-100,000 pmolsTaqMan ® Probes (5’-Fluorescent label: 6-FAM, VIC or TET; 3’-label: TAMRA)450025 5,000-6,000 pmols450024 15,000-25,000 pmols450023 50,000-100,000 pmolsC-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Custom Oligonucleotide SynthesisTable C-4Custom OligonucleotidesPartNumberDescriptionSequence Detection Primers4304970 Minimum 4,000 pmols purified for sequence detection4304971 Minimum 40,000 pmols purified for sequence detection4304972 Minimum 130,000 pmols purified for sequence detectionApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide C-7DRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Appendix CKits, Reagents, and ConsumablesC-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Consumables.fm

Theory of OperationDDIn This Appendix Fluorescent-Based Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2Real-Time Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-4Comparative C T Method of Relative Quantification. . . . . . . . . . . . . . . . . . . . . . . D-8Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-1DRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Appendix DTheory of OperationFluorescent-Based ChemistriesFundamentals ofthe 5´ NucleaseAssayThe PCR reaction exploits the 5´ nuclease activity of AmpliTaq Gold ® DNAPolymerase to cleave a TaqMan ® probe during PCR. The TaqMan probe contains areporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe.During the reaction, cleavage of the probe separates the reporter dye and thequencher dye, which results in increased fluorescence of the reporter. Accumulationof PCR products is detected directly by monitoring the increase in fluorescence ofthe reporter dye. Figure D-1 shows the forklike-structure-dependent,polymerization-associated 5´–3´ nuclease activity of AmpliTaq Gold DNAPolymerase during PCR.PolymerizationFowardPrimerRProbe5'3'3'5'5' 3'Strand displacementRRQR = ReporterQ = QuencherReversePrimer5'Probe3'3'5'5' 3'5'Cleavage5'Probe3'3'5'5' 3'5'Polymerization completedRQQProbe5'3'5'5' 3'5'Q3'5'Figure D-15´–3´ Nuclease Activity of AmpliTaq Gold DNA PolymeraseWhen the probe is intact, the proximity of the reporter dye to the quencher dye resultsin suppression of the reporter fluorescence primarily by Förster-type energy transfer(Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, theprobe specifically anneals between the forward and reverse primer sites.The 5´–3´ nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves theprobe between the reporter and the quencher only if the probe hybridizes to thetarget. The probe fragments are then displaced from the target, and polymerization ofthe strand continues. The 3´ end of the probe is blocked to prevent extension of theprobe during PCR. This process occurs in every cycle and does not interfere with theexponential accumulation of product.The increase in fluorescence signal is detected only if the target sequence iscomplementary to the probe and is amplified during PCR. Because of theserequirements, any nonspecific amplification is not detected.D-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Fluorescent-Based ChemistriesBasics of SYBRGreen DyeChemistryThe SYBR ® Green 1 Double-Stranded Binding Dye is used for the fluorescentdetection of double-stranded DNA (dsDNA) generated during PCR. The SYBRGreen 1 Dye binds non-specifically to dsDNA and generates an excitation-emissionprofile similar to that of the FAM reporter dye. When used in combination with apassive reference, the SYBR Green 1 Dye can be employed to perform severalSDS-related experiments including quantitative PCR and dissociation cure analysis.Figure D-2 illustrates the action of the SYBR Green 1 dye during a single cycle of aPCR.3´5´DissociationWhen added to the reaction, the SYBR Green 1 Dye binds non-specifically tothe hybridized dsDNA and fluoresces5´3´3´5´5´3´PolymerizationDenaturation complete, the SYBR Green 1 Dye dissociates from the strand,resulting in decreased fluorescence5´3´5´Polymerization Complete3´5´3´5´ForwardPrimerReversePrimerDuring the extension phase, the SYBR Green 1 Dye begins binding to thePCR productPolymerization is complete and SYBR Green 1 Dye is completely bound,resulting in a net increase in fluorescence5´3´5´5´3´5´3´Figure D-2Binding Activity of the SYBR Green 1 Dye during the PCRApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-3DRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Appendix DTheory of OperationReal-Time Data AnalysisKinetic Analysis/Quantitative PCRThe 7900HT instrument can be used to determine the absolute or relative quantity ofa target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle changein fluorescence signal as a result of amplification during a PCR. This form ofquantitative PCR analysis, called “kinetic analysis,” was first described using anon-sequence-specific fluorescent dye, ethidium bromide, to detect PCR product(Higuchi et al., 1992; Higuchi et al., 1993). The use of TaqMan probes and reagentsfurther enhances the method by providing sequence-specific amplification ofmultiple targets for ‘comparative’ or ‘relative’ quantification. The fewer cycles ittakes to reach a detectable level of fluorescence, the greater the initial copy numberof the target nucleic acid.Amplification Plot8.02.01.0Phase 3, plateauPhase 2, linearPhase 1, geometricR n0.0010203040Cycle NumberFigure D-3Phases of a Typical Amplification CurveWhen graphed in real-time on a linear scale, normal amplification of PCR productgenerates a curve similar to the one shown in Figure D-3. This ‘amplification’ curveconsists of three distinct regions that characterize the progression of the PCR.Phase 1: Geometric (Exponential)Detection of the high-precision geometric phase is the key to high-precisionquantitative PCR. The geometric phase is a cycle range of high precision duringwhich amplification is characterized by a high and constant efficiency. It occursbetween the first detectable rise in fluorescence and before the beginning of thelinear phase. When plotted on a log scale of DNA vs. cycle number, the curvegenerated by the geometric phase should approximate a straight line with a slope.The 7900HT instrument typically delivers sufficient sensitivity to detect at least3 cycles in the geometric phase, assuming reasonably optimized PCR conditions.D-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Real-Time Data AnalysisPhase 2: LinearThe linear phase is characterized by a leveling effect where the slope of theamplification curve decreases steadily. At this point, one or more components havefallen below a critical concentration and the amplification efficiency has begun todecrease. This phase is termed linear, because amplification approximates anarithmetic progression, rather than a geometric increase. Because the amplificationefficiency is continually decreasing during the Linear phase, it exhibits lowprecision.Phase 3: PlateauFinally, the amplification curve achieves the plateau phase at which time the PCRstops and the R n signal remains relatively constant.DeterminingInitial TemplateConcentrationand CycleNumberAt any given cycle inside the geometric phase of PCR, the amount of product isproportional to the initial number of template copies. When one template is dilutedseveral times, as with the RNase P target in the RNase P Instrument VerificationPlates (see Appendix C), the ratio of template concentration to detectable signal ispreserved in the exponential phase for all dilutions (see Figure D-4). Thisrelationship appears to change as rate of amplification approaches a plateau.8.02.01.02000010000500025001250R n0.0010203040Cycle NumberFigure D-4 Amplification Plot from a Real-Time Run of an RNase P InstrumentVerification PlateApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-5DRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Appendix DTheory of OperationFluorescence vs.AmplifiedProductWhen using TaqMan fluorogenic probes with the 7900HT instrument, fluorescenceemission increases in direct proportion to the amount of specific amplified product.As Figure D-4 on page D-5 demonstrates, the graph of normalized reporter (R n ) vs.cycle number during PCR appears to have three stages. Initially, R n appears as a flatline because the fluorescent signal is below the detection limit of the 7900HTinstrument. In the second stage, the signal can be detected as it continues to increasein direct proportion to the increase in the products of PCR. As PCR productcontinues to increase, the ratio of AmpliTaq Gold polymerase to PCR productdecreases. When template concentration reaches 10 -8 M, PCR product ceases to growexponentially. This signals the third stage of R n change, which is roughly linear andfinally reaches a plateau at about 10 -7 M (Martens and Naes, 1989).The progressive cleavage of TaqMan fluorescent probes during the PCR makespossible the correlation between initial template concentration and the rise influorescence. As the concentration of amplified product increases in a sample, sodoes the R n value. During the exponential growth stage (the geometric phase), therelationship of amplified PCR product to initial template can be shown in thefollowing equation:N c= N1 ( + E) cwhere N c is the concentration of amplified product at any cycle, N is the initialconcentration of target template, E is the efficiency of the system, and c is the cyclenumber.CalculatingThreshold CyclesThe Applied Biosystems 7900HT Fast Real-Time PCR System creates quantifiablerelationships between test samples based on the number of cycles elapsed beforeachieving detectable levels of fluorescence. Test samples containing a greater initialtemplate number cross the detection threshold at a lower cycle than samplescontaining lower initial template. The SDS software uses a Threshold setting todefine the level of detectable fluorescence.The threshold cycle (C T ) for a given amplification curve occurs at the point that thefluorescent signal grows beyond the value of the threshold setting. The C T representsa detection threshold for the 7900HT instrument and is dependent on two factors:• Starting template copy number• Efficiency of DNA amplification the PCR systemHow the SDS Software Determines C T sTo determine the C T for an Amplification plot, the SDS software uses data collecteddata from a predefined range of PCR cycles called the ‘baseline’ (the defaultbaseline occurs between cycles 3 and 15). First, the software calculates amathematical trend based on the baseline cycles’ R n values to generate a baselinesubtracted Amplification plot of ∆R n versus cycle number. Next, an algorithmsearches for the point on the Amplification plot at which the ∆R n value crosses thethreshold setting (the default threshold setting is 0.2). The fractional cycle at whichthe intersection occurs is defined as the threshold cycle (C T ) for the plot.Note: It may be necessary to adjust the baseline and threshold settings to obtainaccurate and precise data. For further information on resetting the baseline andthreshold settings, see “Setting the Baseline and Threshold Values” on page 6-46.D-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

Real-Time Data AnalysisSignificance ofThreshold CyclesBeginning with the equation describing the exponential amplification of the PCR:X n = X m ( 1 + E X ) n – mwhere:X n = number of target molecules at cycle n (so that n ≥ m)X m = number of target molecules at cycle m (so that m ″ n)E X = efficiency of target amplification (between 0–1)n - m = number of cycles elapsed between cycle m and cycle nAmplicons designed and optimized according to Applied Biosystems guidelines(amplicon size

Appendix DTheory of OperationComparative C T Method of Relative QuantificationArithmeticFormulaThe Comparative C T Method is similar to the Standard Curve Method. However, ituses arithmetic formulas to achieve the same results for relative quantification. Theamount of target, normalized to an endogenous reference and relative to a calibrator,is given by:–∆∆C2TDerivation of theFormulaThe equation that describes the exponential amplification of PCR is:X n = X o × ( 1 + E X ) nwhere:X n = number of target molecules at cycle nX o = initial number of target moleculesE X = efficiency of target amplificationn= number of cyclesThe threshold cycle (C T ) indicates the fractional cycle number at which the amountof amplified target reaches a fixed threshold.Thus,CX T = X o × ( 1 + E X ) TX ,= K Xwhere:X T = threshold number of target moleculesC T,X = threshold cycle for target amplificationK X = constantD-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm

A similar equation for the endogenous reference reaction is:CR T = R o × ( 1 + E R ) TR ,= K Rwhere:R T = threshold number of reference moleculesR o = initial number of reference moleculesE R = efficiency of reference amplificationC T, R = threshold cycle for reference amplificationK R = constantDividing X T by R T gives the following expression:X------ TR T=X o ( 1 E X ) C TX ,× +---------------------------------------------R o ( 1 E R ) C =TR ,× +K------- X= KK RThe exact values of X T and R T depend on a number of factors, including:• Reporter dye used in the probe• Sequence context effects on the fluorescence properties of the probe• Efficiency of probe cleavage• Purity of the probe• Setting of the fluorescence thresholdTherefore, the constant K does not have to be equal to one.Assuming efficiencies of the target and the reference are equivalent:E X = E R = E,X oC TX , – C------ TR ,× ( 1 + E ) = KR oor∆X N ( 1 E ) C T× + = Kwhere:X N∆C T= X o /R o , the normalized amount of target= C T, X - C T, R , the difference in threshold cycles for target and reference

Appendix DTheory of OperationRearranging gives the following expression:–∆C TX N = K × ( 1 + E)The final step is to divide the X N for any sample q by the X N for the calibrator (cb):X-------------- N,qX N,cb–∆C Tq ,K × ( 1 + E )–∆∆C T--------------------------------------------- ( 1 + E )–∆C Tcb ,K × ( 1 + E )= =where:∆∆C T= ∆C T,q – ∆C T,cbFor amplicons designed and optimized according to Applied Biosystems guidelines(amplicon size

Limited Warranty StatementEEWarrantyStatementApplera Corporation, through its Applied Biosystems Group(“Applied Biosystems“) warrants to the customer that, for a period ending on theearlier of one year from the completion of installation or fifteen (15) months from thedate of shipment to the customer (the “Warranty Period”), the Applied Biosystems7900HT Fast Real-Time PCR System purchased by the customer (the “Instrument”)will be free from defects in material and workmanship, and will perform inaccordance with the installation specifications set forth in the system specificationssheet which accompanies the instrument or which is otherwise available from anApplied Biosystems sales representative.During the Warranty Period, if the Instrument's hardware becomes damaged orcontaminated or if the Instrument otherwise fails to meet the Specifications,Applied Biosystems will repair or replace the Instrument so that it meets theSpecifications, at Applied Biosystems' expense. However, if the thermal cyclingmodule becomes damaged or contaminated, or if the chemical performance of theInstrument otherwise deteriorates due to solvents and/or reagents other than thosesupplied or expressly recommended by Applied Biosystems, Applied Biosystemswill return the Instrument to Specification at the customer's request and at thecustomer's expense. After this service is performed, coverage of the parts repaired orreplaced will be restored thereafter for the remainder of the original Warranty Period.This Warranty does not extend to any Instrument or part which has been (a) thesubject of an accident, misuse, or neglect, (b) modified or repaired by a party otherthan Applied Biosystems, or (c) used in a manner not in accordance with theinstructions contained in the Instrument User's Manual. This Warranty does notcover the customer-installable accessories or customer-installable consumable partsfor the Instrument that are listed in the Instrument User's Manual. Those items arecovered by their own warranties.Applied Biosystems' obligation under this Warranty is limited to repairs orreplacements that Applied Biosystems deems necessary to correct those failures ofthe Instrument to meet the Specifications of which Applied Biosystems is notifiedprior to expiration of the Warranty Period. All repairs and replacements under thisWarranty will be performed by Applied Biosystems on site at the Customer'slocation at Applied Biosystems's sole expense.No agent, employee, or representative of Applied Biosystems has any authority tobind Applied Biosystems to any affirmation, representation, or warranty concerningthe Instrument that is not contained in Applied Biosystems's printed productliterature or this Warranty Statement. Any such affirmation, representation orwarranty made by any agent, employee, or representative of Applied Biosystems willnot be binding on Applied Biosystems.Applied Biosystems shall not be liable for any incidental, special, or consequentialloss, damage or expense directly or indirectly arising from the purchase or use of theInstrument. Applied Biosystems makes no warranty whatsoever with regard toproducts or parts furnished by third parties.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide E-1DRAFTSeptember 1, 2004 11:38 am, App_Warranty.fm

Appendix ELimited Warranty StatementThis Warranty is limited to the original location of installation and is not transferable.THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THEINSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIEDWARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIEDWARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULARPURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OFAPPLIED BIOSYSTEMS.E-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, App_Warranty.fm

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Klein, D., Janda, P., Steinborn, R., Muller, M., Salmons, B., and Gunzburg, W.H.Proviral load determination of different feline immunodeficiency virus isolates usingreal-time polymerase chain reaction: influence of mismatches on quantification [InProcess Citation]. Electrophoresis 20(2):291-299, 1999.Lo, Y. M., et al. "Quantitative analysis of fetal DNA in maternal plasma and serum:implications for noninvasive prenatal diagnosis." Am J Hum Genet 62 (1998):768-775.Lo, Y.M., Chan, L.Y., Lo, K.W., Leung, S.F., Zhang, J., Chan, A.T., Lee, J.C., Hjelm,N.M., Johnson, P.J., and Huang, D.P. Quantitative analysis of cell-free Epstein-Barrvirus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res.59(6):1188-1191, 1999.Lo, Y.M., Leung, T.N., Tein, M.S., Sargent, I.L., Zhang, J., Lau, T.K., Haines, C.J., andRedman, C.W. Quantitative abnormalities of fetal DNA in maternal serum inpreeclampsia [see comments]. Clin.Chem. 45(2):184-188, 1999.Lo Y.M., L.Y. Chan, A.T. Chan, S.F. Leung, K.W. Lo, J. Zhang, J.C. Lee, N.M. Hjelm,P.J. Johnson and D.P. Huang. 1999. Quantitative and temporal correlation betweencirculating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngealcarcinoma. Cancer Res. 59:5452-5455.Leutenegger, C.M., Klein, D., Hofmann-Lehmann, R., Mislin, C., Hummel, U., Boni,J., Boretti, F., Guenzburg, W.H., and Lutz, H. Rapid feline immunodeficiency virusprovirus quantitation by polymerase chain reaction using the TaqMan fluorogenicreal-time detection system [In Process Citation]. J.Virol.Methods 78(1-2):105-116,1999Lockey, C., E. , Otto, and Z. , Long. "Real-time fluorescence detection of a singleDNA molecule." BioTechniques 24 (1998): 744-746.Luthra, R., et al. "Novel 5' exonuclease-based real-time PCR assay for the detection oft(14;18)(q32;q21) in patients with follicular lymphoma." Am J Pathol 153 (1998):63-68.Maudru, T. and K. W., Peden. "Adaptation of the fluorogenic 5'-nuclease chemistry toa PCR-based reverse transcriptase assay." BioTechniques 25 (1998): 972-975.Nagai, M., Usuku, K., Matsumoto, W., Kodama, D., Takenouchi, N., Moritoyo, T.,Hashiguchi, S., Ichinose, M., Bangham, C.R., Izumo, S., and Osame, M. Analysis ofHTLV-I proviral load in 202 HAM/TSP patients and 243 asymptomatic HTLV-Icarriers: high proviral load strongly predisposes to HAM/TSP. J.Neurovirol.4(6):586-593, 1998.Nogva, H.K. and Lillehaug, D. Detection and quantification of Salmonella in purecultures using 5'-nuclease polymerase chain reaction [In Process Citation]. Int.J.FoodMicrobiol. 51(2-3):191-196, 1999.Pusterla, N., Huder, J.B., Leutenegger, C.M., Braun, U., Madigan, J.E., and Lutz, H.Quantitative real-time PCR for detection of members of the Ehrlichia phagocytophilagenogroup in host animals and Ixodes ricinus ticks. J.Clin.Microbiol.37(5):1329-1331, 1999.Pusterla, N., Leutenegger, C.M., Chae, J.S., Lutz, H., Kimsey, R.B., Dumler, J.S., andMadigan, J.E. Quantitative Evaluation of Ehrlichial Burden in Horses afterExperimental Transmission of Human Granulocytic Ehrlichia Agent by IntravenousInoculation with Infected Leukocytes and by Infected Ticks. J.Clin.Microbiol.37(12):4042-4044, 1999.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-3DRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

RelativeQuantitationSmith, G.J., et al. "Fast and accurate method for quantitating E. coli host-cell DNAcontamination in plasmid DNA preparations." BioTechniques 26 (1999): 518-526.Wang, S.C., Klein, R.D., Wahl, W.L., Alarcon, W.H., Garg, R.J., Remick, D.G., andSu, G.L. Tissue coexpression of LBP and CD14 mRNA in a mouse model of sepsis.J.Surg.Res. 76(1):67-73, 1998.Wang, T. and Brown, M.J. mRNA Quantification by Real Time TaqMan PolymeraseChain Reaction: Validation and Comparison with RNase Protection [In ProcessCitation]. Anal.Biochem. 269(1):198-201, 1999.Wang X., X. Li, J.A. Erhardt, F.C. Barone and G.Z. Feuerstein. 2000. Detection oftumor necrosis factor-alpha mRNA induction in ischemic brain tolerance by means ofreal-time polymerase chain. J. Cereb. Blood Flow Metab. 2000. Jan;20(1):15-20.20:15-20.Wang X., X. Li, R.W. Currie, R.N. Willette, F.C. Barone and G.Z. Feuerstein. 2000.Application of real-time polymerase chain reaction to quantitate induced expression ofinterleukin-1beta mRNA in ischemic brain tolerance. J. Neurosci. Res. 2000. Jan.15;59(2):238-46.Ying H., T.Z. Zaks, R.F. Wang, K.R. Irvine, U.S. Kammula, F.M. Marincola, W.W.Leitner and N.P. Restifo. Cancer therapy using a self-replicating RNA vaccine. Nat.Med. 5:823-827, 1999.Araki, R. and et al Nonsense mutation at Tyr-406 in the DNA-dependent proteinkinase catalytic subunit of severe combined immune deficiency mice. PNAS94:2438-2443, 1997.Bieche, I., et al. "Novel approach to quantitative polymerase chain reaction using realtimedetection: application to the detection of gene amplification in breast cancer." IntJ Cancer 78 (1998): 661-666.Bieche I., P. Onody, I. Laurendeau, M. Olivi, D. Vidaud, R. Lidereau and M. Vidaud.Real-time reverse transcription-PCR assay for future management of ERBB2-basedclinical applications. Clin. Chem. 45:1148-1156, 1999.Casteels, K. M., et al. "Prevention of type I diabetes in nonobese diabetic mice by lateintervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 incombination with a short induction course of cyclosporin A." Endocrinology 139(1998): 95-102.Chiang, P.W., Beer, D.G., Wei, W.L., Orringer, M.B., and Kurnit, D.M. Detection oferbB-2 amplifications in tumors and sera from esophageal carcinoma patients.Clin.Cancer Res. 5(6):1381-1386, 1999.Collins, C., et al. "Positional cloning of ZNF217 and NABC1: genes amplified at20q13.2 and overexpressed in breast carcinoma." Proc Natl Acad Sci U S A 95 (1998):8703-8708.de Kok, J.B., Ruers, T.J., van Muijen, G.N., van Bokhoven, A., Willems, H.L., andSwinkels, D.W. Real-time quantification of human telomerase reverse transcriptasemRNA in tumors and healthy tissues. Clin.Chem.2000.Mar;46(3):313-8.Dittmer D., C. Stoddart, R. Renne, V. Linquist-Stepps, M.E. Moreno, C. Bare, J.M.McCune and D. Ganem. 1999. Experimental Transmission of Kaposi'sSarcoma-associated Herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv Mice. J. Exp.Med. 190:1857-1868.Bibliography-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

Dolken, L., F. , Schuler, and G. , Dolken. "Quantitative detection of t(14;18)-positivecells by real-time quantitative PCR using fluorogenic probes." BioTechniques 25(1998): 1058-1064.Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Danenberg, P.V., and Laird,P.W. CpG island hypermethylation in human colorectal tumors is not associated withDNA methyltransferase overexpression. Cancer Res. 59(10):2302-2306, 1999.Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Blake, C., Shibata, D.,Danenberg, P.V., and Laird, P.W. MethyLight: a high-throughput assay to measureDNA methylation. Nucleic.Acids.Res.2000.Apr.15;28(8):E32.Eder, M., Battmer, K., Kafert, S., Stucki, A., Ganser, A., and Hertenstein, B.Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bonemarrow or peripheral blood stem cell transplantation. Leukemia 13(9):1383-1389,1999.Fink, L., et al. "Real-time quantitative RT-PCR after laser-assisted cell picking." NatMed 4 (1998): 1329-1333.Haugland, R.A., Vesper, S.J., and Wymer, L.J. Quantitative measurement ofstachybotrys chartarum conidia using real time detection of PCR products with theTaqMan(TM)fluorogenic probe system. Mol.Cell Probes. 13(5):329-340, 1999.Hirayama, Y., et al. "Concentrations of thrombopoietin in bone marrow in normalsubjects and in patients with idiopathic thrombocytopenic purpura, aplastic anemia,and essential thrombocythemia correlate with its mRNA expression of bone marrowstromal cells." Blood 92 (1998): 46-52.Johnson, M.R., Wang, K., Smith, J.B., Heslin, M.J., and Diasio, R.B. Quantitation ofdihydropyrimidine dehydrogenase expression by real-time reverse transcriptionpolymerase chain reaction. Anal.Biochem.2000.Feb.15;278(2):175-84.Krauter J., M.P. Wattjes, S. Nagel, O. Heidenreich, U. Krug, S. Kafert, D. Bunjes, L.Bergmann, A. Ganser and G. Heil. 1999. Real-time RT-PCR for the detection andquantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients.Br. J. Haematol. 107:80-85.Kruse, N., Pette, M., Toyka, K., and Rieckmann, P. Quantification of cytokine mRNAexpression by RT PCR in samples of previously frozen blood. J.Immunol.Methods210(2):195-203, 1997.Lee, L.Y., Patel, S.R., Hackett, N.R., Mack, C.A., Polce, D.R., El-Sawy, T.,Hachamovitch, R., Zanzonico, P., Sanborn, T.A., Parikh, M., Isom, O.W., Crystal,R.G., and Rosengart, T.K. Focal angiogen therapy using intramyocardial delivery of anadenovirus vector coding for vascular endothelial growth factor 121.Ann.Thorac.Surg.2000.Jan;69(1):14-23;.discussion.23-4.Leutenegger C.M., C.N. Mislin, B. Sigrist, M.U. Ehrengruber, R. Hofmann-Lehmannand H. Lutz. 1999. Quantitative real-time PCR for the measurement of feline cytokinemRNA. Vet. Immunol. Immunopathol. 71:291-305.Maeda, I., Takano, T., Matsuzuka, F., Maruyama, T., Higashiyama, T., Liu, G., Kuma,K., and Amino, N. Rapid screening of specific changes in mRNA in thyroidcarcinomas by sequence specific-differential display: decreased expression of acidceramidase mRNA in malignant and benign thyroid tumors [In Process Citation].Int.J.Cancer 81(5):700-704, 1999.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-5DRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

Mandigers, C.M., Meijerink, J.P., Raemaekers, J.M., Schattenberg, A.V., and Mensink,E.J. Graft-versus-lymphoma effect of donor leucocyte infusion shown by real- timequantitative PCR analysis of t(14;18) [letter]. Lancet 352(9139):1522-1523, 1998.Marcucci, G., et al. "Detection of minimal residual disease in patients withAML1/ETO-associated acute myeloid leukemia using a novel quantitative reversetranscription polymerase chain reaction assay." Leukemia 12 (1998): 1482-1489.Mensink, E., et al. "Quantitation of minimal residual disease in Philadelphiachromosome positive chronic myeloid leukaemia patients using real-time quantitativeRT-PCR." Br J Haematol 102 (1998): 768-774.Moody, A., Sellers, S., and Bumstead, N. Measuring infectious bursal disease virusRNA in blood by multiplex real-time quantitative RT-PCR. Virol.Methods2000.Mar;85(1-2):55-64.Patterson, B. K., et al. "Repertoire of chemokine receptor expression in the femalegenital tract: implications for human immunodeficiency virus transmission." Am JPathol 153 (1998): 481-490.Pongers-Willemse, M.J., Verhagen, O.J., Tibbe, G.J., Wijkhuijs, A.J., de Haas, V.,Roovers, E., van der Schoot, C.E., and van Dongen, J.J. Real-time quantitative PCRfor the detection of minimal residual disease in acute lymphoblastic leukemia usingjunctional region specific TaqMan probes. Leukemia 12(12):2006-2014, 1998.Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K.A., Boxer, L.M., Heasley,L.E., and Reusch, J.E. Akt/protein kinase B up-regulates Bcl-2 expression throughcAMP-response element-binding protein. J.Biol.Chem.2000.Apr.14;275(15):10761-6.Raggi, C.C., Bagnoni, M.L., Tonini, G.P., Maggi, M., Vona, G., Pinzani, P., Mazzocco,K., De Bernardi, B., Pazzagli, M., and Orlando, C. Real-time quantitative PCR for themeasurement of MYCN amplification in human neuroblastoma with the TaqMandetection system. Clin.Chem. 45(11):1918-1924, 1999.Ringel, M.D., Balducci-Silano, P.L., Anderson, J.S., Spencer, C.A., Silverman, J.,Sparling, Y.H., Francis, G.L., Burman, K.D., Wartofsky, L., Ladenson, P.W., Levine,M.A., and Tuttle, R.M. Quantitative reverse transcription-polymerase chain reaction ofcirculating thyroglobulin messenger ribonucleic acid for monitoring patients withthyroid carcinoma. J.Clin.Endocrinol.Metab. 84(11):4037-4042, 1999.Sangoram, A. M., et al. "Mammalian circadian autoregulatory loop: a timelessortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-inducedtranscription." Neuron 21 (1998): 1101-1113.Sgroi D.C., S. Teng, G. Robinson, R. LeVangie, J.R. Hudson, Jr. and A.G. Elkahloun.1999. In vivo gene expression profile analysis of human breast cancer progression.Cancer Res. 59:5656-5661.Shayesteh, L., Lu, Y., Kuo, W.L., Baldocchi, R., Godfrey, T., Collins, C., Pinkel, D.,Powell, B., Mills, G.B., and Gray, J.W. PIK3CA is implicated as an oncogene inovarian cancer [see comments]. Nat.Genet. 21(1):99-102, 1999.Shimokawa, T., et al. "Transcriptional regulation of muscle-specific genes duringmyoblast differentiation." Biochem Biophys Res Commun 246 (1998): 287-292.Taoufik, Y., Froger, D., Benoliel, S., Wallon, C., Dussaix, E., Delfraissy, J.F., andLantz, O. Quantitative ELISA-polymerase chain reaction at saturation usinghomologous internal DNA standards and chemiluminescence revelation.Eur.Cytokine.Netw. 9(2):197-204, 1998.Bibliography-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

AllelicDiscriminationVirtanen M., H. Torma and A. Vahlquist. 2000. Keratin 4 upregulation by retinoic acidIn vivo: A sensitive marker for retinoid bioactivity in human epidermis. J. Invest.Dermatol. 2000. Mar;114(3):487-93.Wingo, S.T., Ringel, M.D., Anderson, J.S., Patel, A.D., Lukes, Y.D., Djuh, Y.Y.,Solomon, B., Nicholson, D., Balducci-Silano, P.L., Levine, M.A., Francis, G.L., andTuttle, R.M. Quantitative reverse transcription-PCR measurement of thyroglobulinmRNA in peripheral blood of healthy subjects. Clin.Chem. 45(6 Pt 1):785-789, 1999.Yajima, T., et al. "Quantitative reverse transcription-PCR assay of the RNA componentof human telomerase using the TaqMan fluorogenic detection system." ClinicalChemistry 44 (1998): 2441-2445.Yamada Y., N. Itano, H. Narimatsu, T. Kudo, S. Hirohashi, A. Ochiai, A. Niimi, M.Ueda and K. Kimata. 1999. Receptor for hyaluronan-mediated motility and CD44expressions in colon cancer assessed by quantitative analysis using real-time reversetranscriptase-polymerase chain reaction. Jpn. J. Cancer Res. 90:987-992.Yoneyama H., A. Harada, T. Imai, M. Baba, O. Yoshie, Y. Zhang, H. Higashi, M.Murai, H. Asakura and K. Matsushima. 1998. Pivotal role of TARC, a CC chemokine,in bacteria-induced fulminant hepatic failure in mice. J. Clin. Invest. 102:1933-1941.Zhang, A.W., Hartman, G.L., Curio-Penny, B., Pedersen, W.L., and Becker, K.B.Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla fromSoybean Seeds. Phytopathology 89(9):796-804, 1999.Breen, G., Harold, D., Ralston, S., Shaw, D., and St Clair, D. Determining SNP allelefrequencies in DNA pools. Biotechniques 2000.Mar;28(3):464-8,.470.Fujii K., Y. Matsubara, J. Akanuma, K. Takahashi, S. Kure, Y. Suzuki, M. Imaizumi,K. Iinuma, O. Sakatsume, P. Rinaldo and K. Narisawa. 2000. Mutation detection byTaqMan-allele specific amplification: application to molecular diagnosis of glycogenstorage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency. Hum.Mutat. 2000;15(2):189-96.Germer, S. and Higuchi, R. Single-tube genotyping without oligonucleotide probes.Genome Res. 9(1):72-78, 1999.Happich, D., Schwaab, R., Hanfland, P., and Hoernschemeyer, D. Allelicdiscrimination of factor V Leiden using a 5' nuclease assay. Thromb.Haemost.82(4):1294-1296, 1999.Holloway, J.W., Beghe, B., Turner, S., Hinks, L.J., Day, I.N., and Howell, W.M.Comparison of three methods for single nucleotide polymorphism typing for DNAbank studies: sequence-specific oligonucleotide probe hybridisation, TaqMan liquidphase hybridisation, and microplate array diagonal gel electrophoresis (MADGE).Hum.Mutat. 14(4):340-347, 1999.Livak, K.J. Allelic discrimination using fluorogenic probes and the 5' nuclease assay[In Process Citation]. Genet.Anal. 14(5-6):143-149, 1999.Mizugaki M., M. Hiratsuka, Y. Agatsuma, Y. Matsubara, K. Fujii, S. Kure and K.Narisawa. 2000. Rapid detection of CYP2C18 genotypes by real-time fluorescencepolymerase chain reaction. J. Pharm. Pharmacol. 2000. Feb;52(2):199-205.*Paris, P.L., Kupelian, P.A., Hall, J.M., Williams, T.L., Levin, H., Klein, E.A., Casey,G., and Witte, J.S. Association between a CYP3A4 genetic variant and clinicalpresentation in African-American prostate cancer patients [In Process Citation].Cancer Epidemiol.Biomarkers.Prev. 8(10):901-905, 1999.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-7DRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

Shi M.M., S.P. Myrand, M.R. Bleavins and F.A. de la Iglesia. High-throughputgenotyping method for glutathione S-transferase T1 and M1 gene deletions usingTaqMan probes. Res. Commun. Mol. Pathol. Pharmacol. 103:3-15, 1999.Bibliography-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, Bibliography.fm

Glossary.sdd.sdm.sds.sdt∆C T∆∆C T∆R nThe file extension of an ABI PRISM SDS Detector file for SDS software for the7900HT instrument.The file extension of a SDS 7900HT Multiple Plate Document (*.sdm) for SDSsoftware for the 7900HT instrument.The file extension of a SDS 7900HT Document (*.sds) for SDS software for the7900HT instrument.The file extension of a SDS 7900HT Template Document (*.sdt) for SDS software forthe 7900HT instrument.The difference between the threshold cycle of a sample assay and the threshold cycleof the corresponding endogenous reference: ∆C T = C T (target) - C T - C T (endogenouscontrol).For a given cDNA target, the difference between the average ∆C T value of a targetsample and the average ∆C T for the corresponding calibrator sample:∆∆C T (test sample) = Avg∆C T (test sample) - Avg∆C T (calibrator sample). This valueis used to calculate expression fold value by the equation: Expression fold value =2 −∆∆C TThe normalized reporter signal minus the baseline signal. The baseline is establishedin the first few cycles of PCR. Like R n , ∆R n is not a fixed value but increases duringPCR when the amplicon copy number increases until the reaction approaches aplateau.5′ nuclease assay The 5′ (five prime) nuclease assay in combination with the TaqMan ® probes serves asthe basis for real-time polymerase chain reaction (PCR) technology on the 7900HTinstrument. See “Fundamentals of the 5´ Nuclease Assay” on page D-2 for a detaileddescription of the reaction.ABI PRISM SDSDetector file (*.sdd)SDS 7900HTMultiple PlateDocument (*.sdm)The SDS detector file is a tab-delimited flat file that is used to transfer detectorinformation to and from the SDS software using the Export and Import functions ofthe Detector Manager software. The file contains: (a) a header“SDS2.2.1[GDF[Global Detector File”, (b) tab-delimited data for each detector (alleleName, the name of the reporter dye, the name of the quencher dye, a description, acomment, the owner, the creation date and time, the modification date and time, andthe color used to code the detector in graphical displays of the SDS software).Multiple plate documents are used for the analysis of relative quantification data.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-1DRAFTSeptember 1, 2004 11:39 am, Glossary.fm

SDS 7900HTDocument (*.sds)SDS 7900HTTemplateDocument (*.sdt)absolutequantification (AQ)alleleallelicdiscrimination (AD)ampliconAmplification plotanalysis sessionannealingApplicationProgram Interface(API)assay typeauto incrementvalueA file used by the SDS software to store plate documentation locally on a computerhard drive. A plate document is a virtual representation of a consumable (plate) usedto contain samples and reagents during a sequence detection run. During the run, thesoftware uses the plate document to record the operation of the instrument (thermalcycling and data collection parameters), organize and store the data gathered duringthe PCR process, store the of the run data, and record analysis and display options.Plate document templates have the same format as the SDS 7900HT Documents(*.sds) but do not store raw data or bar codes.The objective of an absolute quantification experiment is to accurately determine theabsolute quantity of a single nucleic acid target sequence within an unknown sample.The results of an absolute quantification experiment are reported in the same units ofmeasure as the standard used to make them.One of the variant forms of a gene at a particular locus or location on a chromosomeThe process by which two variants of a single nucleic acid sequence present in apopulation of individuals' DNA samples is determined. Single nucleotidepolymorphisms (SNPs) comprise the most common variants that are assayed usingTaqMan reagents.A PCR amplified section of DNAThe Amplification plot displays data from real-time PCR for gene expression assaysafter signal normalization and multi-component analysis. The Amplification plotappears only in plate documents containing analyzed data from real-time runs. It isa 2D plot of normalized reporter fluorescence (for ∆R n ) values versus the cyclenumber. The ∆R n versus cycle Amplification plot is used for calculation thresholdcycle (C T ).A set of analysis results derived from a single raw data set associated with a singleplate document analyzed by the SDS software. The analysis session is persisted in thedatabase and can be retrieved by the SDS software. Multiple analysis sessions can becreated from the same plate document, however each analysis session must have aunique name. An analysis session does not have to be associated with a Study;however, if it has such a relationship, it can be associated with at most one Study atany given time.Binding a probe to the DNA or cDNA during the PCR processA set of routines, protocols, and tools for building software applications. A good APImakes it easier for a programmer to develop a program by providing all the buildingblocks necessary to link the elements of the various subcomponents of the system.With reference to the SDS 2.2 Enterprise Option software, an assay type is one of thefollowing: allelic discrimination, absolute quantification, or relative quantification.Method setting that increases or decreases the value for any PCR set-point parameter(time or temperature) by a fixed amount every cycle.Glossary-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, Glossary.fm

ackgroundbaselinecalibratorcDNA(complementaryDNA)clipped datacommand lineconsumablecontainer typecycle set-pointAn inherent spectral component of the signal generated by a fluorescence assay thatis attributed to the Applied Biosystems 7900HT Fast Real-Time PCR System.Uncorrected background signal can interfere with the sensitivity of the SDS softwareand its ability to determine C T s. For this reason background signal is measured duringcalibration and is removed mathematically during multicompoenting.In terms of the Amplification plot, the baseline is a line fit to defined range of cyclesbefore the sequence detection system detects the amplification of the PCR product.For standard PCR assays, the SDS software uses a default range of cycles 3-15 toestablish the baseline for a PCR assay.In relative quantification analysis a sample to which all other samples are referencedon a detector-by-detector basis.A DNA molecule with a nucleotide sequence that is complementary to an RNAmolecule. cDNA is made from RNA through the action of the enzyme ReverseTranscriptase. RNA is converted to cDNA because cDNA is more robust than fragileRNA, and because DNA templates are required for many applications, including PCR.In real time mode, fluorescent signal intensity is collected for the entire plate every8.5 seconds. Clipped data points are calculated by averaging the last three computedR n (normalized reporter signal) data points collected during the extension phase ofeach cycle repetition. Clipped data are presented as baseline uncompensated data(R n vs. cycle data) and baseline compensated data (∆R n vs. cycle data).Some executable programs can be invoked in a DOS Command Prompt windowand/or from within a.bat script file, to run usually without a graphic user interface(GUI). Such programs may support user specification of parameters via a commandline, where those parameters would be entered after the name of the executableprogram. Often these command line parameters are filenames, directory paths, or flagsettings.(1) A vessel used to contain reactions run on the 7900HT instrument (a microplate ora TaqMan ® Low Density Array). (2) A reagent kit, chemical, or disposable labwareused as part of an assay.The type of plate to be analyzed by the 7900HT instrument. The types are:ABI PRISM 96-Well Optical Reaction Plates, ABI PRISM 384-Well OpticalReaction Plates, Optical 96-Well Fast Thermal Cycling Plates, and TaqMan ® LowDensity Arrays.(1) A cycle set-point is any temperature desired for a sample block or heated cover.(2) In the context of thermal cycling, cycle set-point could represent one or moretemperatures in a step or stage.delta C T See ∆C T .delta delta C T See ∆∆C T .delta R n See ∆R n .Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-3DRAFTSeptember 1, 2004 11:39 am, Glossary.fm

detectorendogenouscontrolendogenousreferenceend-point(plate read)run/analysisexpression foldvaluehands-offautomationhigh throughput(HT)holdlocusmarkermethodA virtual representation of a gene- or allele-specific nucleic acid probe reagent usedfor absolute quantification, relative quantification, allelic discrimination, or otheranalyses performed on an SDS instrument. Examples of reagents represented asdetectors include TaqMan probes and the SYBR Green 1 dsDNA Binding Dye.Detector for a gene presented at a consistent level in all experimental samples. Byusing an endogenous control as an active reference, one can normalize quantificationof a messenger RNA (mRNA) target for differences in the amount of total RNA addedto each reaction.See endogenous control.Refers to processing of samples for allelic discrimination and plus/minus scoringchemistries of samples by the 7900HT instrument. The 7900HT instrument collects asingle reading after prior PCR temperature cycles are completed using a thermalcycler. After analysis by the SDS software, the resulting multi-component data is usedto assess the presence of target sequences in the unknown samples.The expression of a target gene relative to control gene in one sample, relative to theexpression level in a calibrator sample. See ∆∆C T .After plates have been loaded into the Zymark ® Twister Microplate Handler stacks,and the Automation Controller Software has been told to start running, no humanintervention should normally be required until all plates in the robot stacks have beenprocessed. With the SDS Enterprise Database option, this includes plate documentanalysis and analysis results persistence to the database.Describes the ability to analyze a large quantity of samples for genotyping or geneexpression analysis.A thermal cycling set-point of a method that maintains a specified temperature for anextended period of time.The physical location of a site on a chromosome, sometimes understood as the site ofa gene, usually understood as the site of a marker variant.For SDS software, a virtual representation of a variable segment of DNA (such as aSNP) whose inheritance can be followed, comprising a name and a set of two detectorsspecific for the allelic variants. Markers are used only for allelic discriminationassays, not for absolute quantification, or relative quantification. Allelicdiscrimination plate documents must contain at least one marker. The marker must beconfigured with two detectors before it can be applied to a plate document.The protocol that the Applied Biosystems 7900HT Fast Real-Time PCR Systemfollows during a real-time or end-point sequence detection run. Methods used by the7900HT instrument make use of the following information: thermal cycling times andtemperatures, ramp rates, auto-increment values, and data collection settings.Glossary-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, Glossary.fm

minor groovebinder (MGB)multicomponentingmulti-reporter(multiplex) PCRNo TemplateControl (NTC)normalized reportersignal (R n )nucleotideoutlierPCR master mixpersist(ed)pure dyeOne of a class of molecules which form close atomic contacts in the deep, narrowspace formed between the two phosphate-sugar backbones in the DNA double helix.Conjugation of a minor groove binder to oligonucleotides stabilizes nucleic acidduplexes causing a dramatic increase in oligonucleotide T m . Fluorogenic probes withthe MGB attached to the 3′ end perform well in the 5′ nuclease assay. They are animprovement over unmodified probes because shorter sequences (13- to 20-mers) canbe used to obtain probes that have an optimal T m . Thus, attachment of the MGBenables the use of shorter fluorogenic probes, which results in improved mismatchdiscrimination.The analysis performed by the SDS software used to distinguish the contribution ofindividual dyes to the raw spectral data collected by the 7900HT instrument. Theoverlapping spectra from the individual dye components in a well generate thecomposite spectrum that represents a raw data fluorescent reading.A technique that uses multiple fluorogenic dye-labeled TaqMan ® probes to detect thesimultaneous amplification of two or more target nucleic acids within a DNA sample.A control reaction with all PCR components included except DNA template. After adetector is applied to a well, each detector must be assigned a task that describes itspurpose. One of the tasks is NTC and is applied to all detectors of negative controlwells containing PCR reagents but lacking samples.See R n .One of the structural components, or building blocks, of DNA and RNA. A nucleotideconsists of a base (one of four chemicals: adenine, thymine, guanine, and cytosine)plus a molecule of sugar and one of phosphoric acid.An unusually extreme value for a variable, given the statistical model in use. In theSDS software, automatic outlier removal in RQ Studies is conducted to assist in theelimination of data contamination occurring when a process or phenomenon otherthan initial sample quantity affects the measured C T .A reagent mix containing all components necessary for PCR except template DNAand primers/probes. For example, the TaqMan ® 2✕ Universal PCR Master Mixcontains AmpliTaq Gold ® DNA polymerase, AmpErase ® UNG that protects againstcarry-over contamination, Passive Reference I for signal normalization in all 5′nuclease assays, dNTPs and optimized buffer components.Stored in, and thus retrievable from, the database used by the SDS software.Pure dye data is generated from the results of a pure dye run in which the SDSsoftware collects spectral data from a set of dye standards during a 2-min. hold at60 °C. The software stores the spectral information for the pure dye standards withina calibration file located in the SDS directory. This calibration file is used forcalculating multi-component data. After the run, the software extracts eachcomponent dye spectrum from the collected data in the pure spectra run file. Becausethe age and use of the instrument components can affect the pure spectra readings,Applied Biosystems recommends updating the pure spectra data files once or twiceannually depending on instrument use.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-5DRAFTSeptember 1, 2004 11:39 am, Glossary.fm

quality valuequantityquantity meanquantity standarddeviationquencher dyeramp(temperature ramp)raw datareaction devicereal-timerun/analysisrelativequantificationreporter dyeThe quality value for a given point is an estimate of the probability that the assignedgenotype for that point is correct, with respect to the rest of the points in the data set.The term quality value is only used for allelic discrimination autocalling.(1) the quantity values for the standards contained on a plate (2) the initial samplequantity computed for the unknowns contained on a plate.Statistical mean of the normally distributed quantity values for a replicate group(wells with the same sample name, detector, and task). Calculated as the sum of allquantity observations (readings) in the replicate group divided by the number ofreplicates.Statistical standard deviation of the normally distributed quantity values for a replicategroup (wells with the same sample name, detector, and task). The standard deviationis calculated using the nonbiased or “n-1” method (square root of sum of the squarednormed residuals divided by n-1 degrees of freedom, with n being the number ofreplicates.The fluorescent dye (or non-fluorescent) banned to the 3′ end of the TaqMan ® Probe,which reduces fluorescence of the reporter dye at the 5′ end by FRET until the reporterdye is cleared during PCR.The interval between two set-points during which the instrument heats or cools thesamples.Unmodified spectral data detected between the wavelengths of 500 nm and 660 nmcollected by the 7900HT instrument during a real-time or end-point run.The physical device used to hold the PCR reactions during evaluation on theApplied Biosystems 7900HT Fast Real-Time PCR System. Examples of reactionformats include ABI PRISM 384- and 96-Well Optical Reaction Plates or theTaqMan ® Low Density Array.Refers to real-time PCR processing of individual plates by the 7900HT instrument forabsolute and relative quantification. The 7900HT instrument collects fluorescencedata during each cycle of a pre-programmed PCR run. The pre-programming occursby setting the thermal cycling parameters of a default method. The step parameters(time and temperature) are adjusted, hold, cycle set, or step are selected, andoptionally, auto increment values, ramp rate values and data collection options can beconfigured.A process by which the quantity of a nucleic acid target, relative to a control target, inone sample is calculated relative to the quantity in another sample. For example, thequantity of c-myc in a brain tissue sample relative to the quantity of c-myc in livertissue sample.Fluorescent dye used to bind to the 5′ end of the TaqMan ® Probe. There are a numberof dyes, such as FAM and VIC ® that covalently bind to the 5′ end of the probe.Glossary-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, Glossary.fm

R nrunsessionsingle nucleotidepolymorphism(SNP)singleplexSNPspectral binspectral calibrationspectral crosstalkstackstudytaskThe normalized reporter signal is the cycle-by-cycle ratio of the fluorescence of thereporter dye and the fluorescence of the passive reference dye in a given well. DuringPCR, R n increases as the amplification copy number increases until the reactionapproaches a plateau. When using the instrument in its Plate Read mode (end pointanalysis), the fluorescence signal is read at a single point in time after the completionof PCR rather than at intervals during the course of PCR.A run describes a collection of fluorescence data carried out by the 7900 instrumentduring and/or after cycling of samples in an optical plate or TaqMan ® Low DensityArray.See Analysis Session.A single base pair of DNA in the genome that differs between individuals.A PCR reaction with a single fluorescent detector per well.See single nucleotide polymorphism.A portion of the complete range of the wave length measured by the 7900HTinstrument (approximately 500-660 nm).Spectral calibration occurs from pure dye results obtained from a pure dye run. Thesoftware stores the spectral information for the pure dye standards within a calibrationfile located in the SDS directory. After a run, the software extracts each componentdye from the collected data in the pure spectra run file. Because the age and use of theinstrument components can affect pure spectra readings, Applied Biosystemsrecommends updating the pure spectra run file once or twice annually depending oninstrument use.The fluorescent contribution of one dye signal to another caused by the overlap of theirspectra.The physical container of a 7900HT instrument into which well plates can be stacked.There are 5 stacks in all. Four of the five consecutively numbered stacks are used tohold the sample plates while the fifth is used by the Plate Handler to hold the first setof analyzed plates. Maximally 20 plates can be placed in each stack. The instrumentcan process maximally eighty plates in one batch.A study in the SDS Enterprise Database option is a named collection of zero or moreAD or RQ analysis sessions. A study in the SDS software main application is an '.sdm'plate document file that contains one or more '.sds' relative quantification platedocument files.A setting applied to the detectors within a well of a plate document that determineshow the data collected from the well during analysis are used. The task selectionsavailable differ depending on the plate's assay type.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-7DRAFTSeptember 1, 2004 11:39 am, Glossary.fm

templatethresholdthreshold cycle (C T )T m(1) [SDS software: SDS 7900HT Template Document or plate document template] Apattern for creating an SDS 7900HT Document (plate document). The filenameextension for a plate document template is *.sdt. The plate document templatefacilitates creation of plate documents in the SDS software. It is typically a startingpoint for batch document setup. The binary representation of the plate documenttemplate is persisted in the database, allowing the plate document template to beretrieved and then populated with data and saved as an SDS 7900HT Document.(2) [Chemical] the DNA sample that is being amplified during PCR.An arbitrary level of normalized reporter signal (R n ) for C T determination in real-timeassays. The level is set to be above the baseline and sufficiently low to be within theexponential growth region of the amplification region of an amplification curve. Thethreshold is the line whose intersection with the Amplification plot defines the C T .For a given well, the threshold cycle (C T ) represents the PCR cycle at which the SDSsoftware first detects a noticeable increase in reporter fluorescence above a baselinesignal.(1)[Chemical] The temperature at which 50% of the DNA amplicons are in adouble-stranded configuration. (2) [Mathematical] The maximum value for the firstderivative curve of normalized reporter fluorescence (R n ).Glossary-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, Glossary.fm

IndexSymbols– 6-31↑ 6-33Numerics7900HT instrument, See instrument9600 emulation mode 3-21AABI PRISM 7700 Sequence Detection System,emulating 3-21ABI PRISM Sealer, see Sealerabsolute quantificationabout 6-6 to 6-7analyzing data 6-9 to 6-13assay development guidelines B-6procedure checklist 6-9selecting and preparing standards B-6troubleshooting 8-12addingbar code to a plate document 2-27, 3-28custom dyes to the pure dye set 7-27detector tasks to a plate document 3-16detectors to a plate document 3-13markers to a plate document 3-15plate documents to the plate queue 4-35, 4-40sample names to a plate document 3-28adjustinganalysis options for absolute quantification 6-11display settings 3-24method step parameters 3-21plate-sensor switch 7-37 to 7-40adjustment knob 7-36air bubbles 8-6aligningfixed-position bar code reader 7-49 to 7-51Plate Handler 7-41 to 7-48allele callsabout 5-15calling 5-14scrutinizing 5-16allelic discriminationabout 5-6 to 5-8analyzing run data 5-10 to 5-18assay development guidelines B-5maximizing throughput 3-4procedure checklist 5-10thermal cycling on the 7900HT instrument 3-20troubleshooting 8-13Allelic Discrimination Plotabout 5-13calling alleles 5-14datapoint cluster variations 5-8exporting as a graphic file A-16exporting data as a text file A-16genotypic segregation 5-8outliers 5-8scrutinizing allele calls 5-16amplificationbeyond reproducible limits in multi-reporterexperiments 6-19amplification curveabout D-4Geometric (Exponential) Phase D-4Linear Phase D-5Plateau Phase D-5Amplification Plotexporting as a graphic file A-16exporting data as a text file A-16Analysis 6-22analysis sessions, about 1-28analyzingabsolute quantification data 6-9 to 6-13allelic discrimination data 5-10 to 5-18background data 7-19dissociation curve data 6-40 to 6-44pure dye data 7-25relative quantification data 6-22 to 6-35applyingdetector tasks 3-16detectors to a plate document 3-13markers to a plate document 3-15sample names 3-28architecturedatabase LAN setup 1-23database WAN setup 1-24Archiver 1-26archiving files 7-54arrows, sample bars 6-31assay development guidelinesabsolute quantification B-2 to B-4, B-6allelic discrimination B-2 to B-5Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideIndex-1DRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

elative quantification B-6automatic outlier removal 6-19Automation Accessorycomponents 7-36Plate Handler, See Plate HandlerSee fixed-position bar code readerAutomation Controller Softwareabout 4-39adding plates to the plate queue 4-40configuring for operation 4-42ejecting a plate 4-44launching 4-39monitoring instrument progress 4-44removing plates from the plate queue 4-40starting the plate queue 4-43stopping the plate queue 4-44Bbackground run 7-16 to 7-19about 7-16constructing a background plate 7-17creating a plate document 7-17extracting 7-19preparing a background plate 7-17troubleshooting 8-9when to perform 7-16bar code informationentering into a plate document 2-27, 3-28bar code readersfixed position, See fixed-position bar code readerhand held, See hand-held bar code readerbars, in the Gene Expression Profilesymbols 6-31baselineabout D-6configuring value for automatic analysis 6-11Block readout (from the Real Time tab) 4-27Bucket Type, setting on centrifuge 4-13Ccalibrating the 7900HT instrumentadjusting the plate-sensor switch 7-37aligning the fixed-position bar code reader 7-49aligning the Plate Handler 7-41performing background run 7-16performing pure dye (spectral) run 7-20calibrator sample 6-20Centrifuge systembucket requirements 4-16Bucket Type, setting 4-13components 4-12installation and operation 4-13placing bucket in centrifuge 4-17requirements 4-12TaqMan Low Density Array, placing inbucket 4-16Centrifuging, TaqMan Low Density Array 4-16changinggripper finger pads 7-52pane, view, and plot sizes 2-21sample block module 7-6checklistsabsolute quantification 6-9allelic discrimination 5-10dissociation curve 6-22, 6-40relative quantification 6-22chemistry5´ Nuclease (TaqMan) D-2non-optimized 8-6SYBR Green D-3troubleshooting 8-5 to 8-8cleaninggripper finger pads 7-52sample block wells 7-15closinginstrument tray (Automation ControllerSoftware) 4-44instrument tray (SDS software) 4-29plate documents 2-18Zymark Twister Software 7-48commentsadding to a detector 3-11adding to a plate document 3-28Comparative C T Methodabout 6-4, D-8formula derivation D-8Componentscentrifuge system 4-12TaqMan Low Density Array 4-10computerabout 1-6hard drive partitions 1-6maintaining 7-54minimum system requirements 1-6troubleshooting 8-14turning ON 2-4configuring the 7900HT instrument for 9600emulation 3-21consumables384-Well Optical Reaction Plates C-396-Well Optical Reaction Plates C-4improper or damaged plastics 8-8Optical 96-Well Fast Thermal Cycling Plates C-4Optical Adhesive Covers C-3Optical Cap Strips C-3writing on reaction plates 8-7Contaminationgenomic DNA 4-11preventing 4-6contaminationdecontaminating the sample block 7-14Index-2Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

fluorescent, common sources 8-7isolating on the sample block module 8-9contextual menus, using 2-28Control assay 4-11copyingdetectors to a plate document 3-13markers to a plate document 3-15Cover readout (from the Real Time tab) 4-27creatingbackground plate documents 7-17custom pure dye plate documents 7-28detectors 3-11markers 3-14plate documents 2-13, 3-9plate documents from a template 3-26, 4-36pure dye plate documents 7-22C T , See threshold cyclecustom pure dyesadding to the pure dye set 7-27 to 7-29creating a custom pure dye plate 7-27cycle set, adding to a method 3-21Ddata type definitions (exportable) A-17databasesaving plate documents 4-24saving results 5-19, 6-53saving templates 3-25database connection A-22decontaminating the sample block module 7-14 to 7-15defragmenting the hard drive 7-54deleting, steps from a method 3-21detector tasksabout 3-16applying 3-16importing into a plate document A-2detectorsabout 3-11applying to a plate document 3-13copying to a plate document 3-13creating 3-11importing into a plate document A-2tasks, See detector tasksdetermining melting temperatures 6-44diagramdatabase LAN setup 1-23database WAN setup 1-24disconnecting the SDS software 4-30display settings, adjusting 3-24dissociation curveabout 6-38analyzing 6-40 to 6-44definitions of the T m value 6-43procedure checklist 6-22, 6-40programming a temperature ramp 3-23Dissociation Plotabout 6-42exporting as a graphic file A-16exporting data as a text file A-16Eejecting a plate from the 7900HT instrument 4-29,4-44endogenous control 6-20end-point runsabout 5-2allelic discrimination, See allelic discriminationenteringbar code information 2-27, 3-28, 4-36comments 3-11, 3-28sample names into a plate document 3-28Equipment needed 4-12exportable data type definitions A-17exportingdata from a plate document 2-14, A-16plots and views as graphic files A-16External endogenous control assay 4-11Ffinger padscleaning 7-52replacing 7-52fixed-position bar code readeraligning 7-49 to 7-51connections 1-10location 1-7specification 1-7fluorescentcontamination 8-7detection system 1-10fluorogenic probeabout D-2designing B-2 to B-3Ggene expression levelmaximum 6-33minimum 6-33gene expression levels 6-31Gene Expression Plot 6-31Genomic DNA contamination, minimizing 4-11genotypic segregation of datapoints(Allelic Discrimination Plot) 5-8Geometric (Exponential) Phase D-4graph, Gene Expression Profilesample bars 6-31X-axis 6-31Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideIndex-3DRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

Y-axis 6-31grey dividing line, using 2-21grid, See plate gridgripper 7-36guidelinesabsolute quantification assay development B-6allelic discrimination assay development B-5loading the Plate Handler 4-41relative quantification assay development B-6TaqMan probe design B-2 to B-3Hhand-held bar code readerconnections 1-10location 1-7specification 1-7using 2-27hard drivedefragmenting 7-54partitions 1-6heated clamp 1-4helpbackground information 2-3using the Sequence Detection Systems SoftwareOnline Help 2-3hold, adding to a method 3-21hotkey combinations, using 2-28Iimporting setup table data 2-16, A-2imprecise pipetting 8-6improper threshold setting 8-5installingplate adapter 7-12sample block module 7-6SDS software 7-55the operating system software 7-55instrument 1-4about 1-2 to 1-10external components 1-3firmware 1-14internal components 1-4loading plates 4-25maintaining 7-4optics system 1-5status lights 2-5supported runs and chemistries 1-2troubleshooting 8-14turning ON 2-4instrument tray 4-44opening and closing (Automation ControllerSoftware) 4-44opening and closing (SDS software) 4-29replacing the plate adapter 7-12IP Address A-22irreproducibilitycauses 8-5 to 8-8JJava Runtime Environment, about 1-14Kkeyboard shortcuts, using 2-28LlaunchingAutomation Controller Software 4-39SDS software 2-7LAVA softwareabout 1-14aligning the fixed-position bar code reader7-51launching 7-49LDHost software, See LAVA softwarelearning to use the SDS software 2-10lights, See instrument, status lightslimited warranty statement E-1Linear Phase of the amplification curve D-5loading platesinto the instrument 4-25into the Plate Handler stacks 4-42low copy templates 8-8M7-49 tomaintenance schedule 7-4managing SDS data 1-17markersabout 3-14applying to a plate document 3-15copying to a plate document 3-15creating 3-14importing into a plate document A-2master mixespreparing B-3using 8-6maximizing, instrument throughput 3-4maximizing/minimizing panes, views, and plots 2-22maximumgene expression level 6-33melting temperaturedefinition 6-43determining 6-44methodsabout 3-19 to 3-23adding a hold, cycle set, or step 3-21adding a temperature ramp 3-23Index-4Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

adjusting step parameters 3-21configuring data collection options 3-22programming 3-20 to 3-23removing a step 3-21setting the sample volume 3-22minimumgene expression level 6-33modes of operation 1-17automated 1-18database 1-18stand-alone 1-17mRNA targets, number per well 4-10Nno template control (NTC)detector task 3-16NTC calls applying 5-15NTC calls verifying 5-16Number of targets per well 4-10O↓ 6-33openinginstrument tray (Automation ControllerSoftware) 4-44instrument tray (SDS software) 4-29plate documents 2-19operating systemsupported 1-6upgrading 7-55operating the 7900HT instrumentpower switch 2-4optimizing primer and probe concentrations B-3outliersallelic discrimination 5-8automatic outlier removal 6-19outlying amplificationmulti-reporter experiments 6-19Ppaneshiding 2-21maximizing/minimizing 2-22resizing 2-21, 2-22showing 2-21passive referencesetting 3-18PCR5´ Nuclease Assay D-2Geometric (Exponential) Phase D-4kinetic analysis of D-4Linear Phase D-5Plateau Phase D-5SYBR Green Chemistry D-3PCR stepfill reservoirs, checking after centrifuging 4-18fill reservoirs, trimming after sealing 4-21general practices 4-6placing in bucket in centrifuge 4-17TaqMan Low Density Array, centrifuging 4-16TaqMan Low Density Array, loading 4-14TaqMan Low Density Array, placing inbucket 4-16TaqMan Low Density Array, sealing 4-19persisted dataanalysis sessions 1-28plate documents1-28studies 1-28pipetting errors 8-6pipettors, using 8-6plate adapter, changing 7-12plate document informationapplying to a plate document 3-28plate documents 3-9about 2-12adding to the plate queue 4-35, 4-40applying detector tasks 3-16applying sample names 3-28assigning standard quantities 3-17closing 2-18configuring document information 3-28copying detectors 3-13copying markers 3-15creating 2-13, 3-9creating from a template 3-26, 4-36exporting 2-14importing setup data 2-16, A-2methods, See methodsopening 2-19programming methods 3-19removing from the plate queue 4-40running batches 4-39running individually 3-29saving 2-17saving as a single plate file 4-24saving as a template file 3-24saving to the database 4-24setting Sample Volume 3-22setting the passive reference 3-18plate gridabout 2-29selecting wells 2-24viewing well information 2-23zooming 2-26Plate Handler 1-8, 7-36adjustment knob 7-36aligning 7-41 to 7-48cleaning the finger pads 7-52gripper 7-36plate stack positions 7-36Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideIndex-5DRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

plate-sensor switch 7-36replacing the finger pads 7-52turning ON 2-4plate queueadding plate documents (Automation ControllerSoftware) 4-40adding plate documents (SDS software) 4-35adding plate documents (Template Batchutility) 4-36removing plate documents 4-40starting 4-43stopping 4-44plate stacksloading plates 4-41, 4-42placing in use 4-42positions 7-36Plateau Phase of the amplification curve D-5platesrunning batches 4-39, 4-41running individually 4-25types, See consumablesplate-sensor switch 7-36adjusting 7-37 to 7-40plotshiding 2-21maximizing/minimizing 2-22resizing 2-21, 2-22showing 2-21Port Number A-22Post readout (from the Plate Read tab) 4-27Pre readout (from the Plate Read tab) 4-27precisioncauses of low precision 8-5 to 8-8preparingmaster mixes B-3plate for a run 4-6primer and probe concentrationsoptimizing B-3programmingmethods (absolute quantification) 3-21methods (allelic discrimination) 3-20methods (dissociation curve analysis) 3-23methods (relative quantification) 3-21temperature ramp 3-23Protocolpreventing contamination 4-6pure dye plateconstructing for custom dyes 7-27preparing for use 7-24pure dye runabout 7-20creating a plate document 7-21extracting data 7-25performing 7-24troubleshooting 8-11when to perform 7-20QQuality control, TaqMan Low Density Array 4-12quantifyingprobes and primers B-3standards for absolute quantification B-6quantitative RT-PCRabout 6-4absolute, See absolute quantificationrelative, See relative quantificationtypes of 6-4quantities, applying to a plate document 3-17RR2 readout (from the Standard Curve Plot) 6-13reagentscustom oligonucleotides C-6non-Applied Biosystems PCR reagents 8-8TaqMan Pre-Developed Assays and Reagents C-6TaqMan RNase P Instrument VerificationPlates C-5re-connecting the SDS software 4-30relative quantificationabout 6-4, 6-16 to 6-20analyzing data 6-22 to 6-35assay development guidelines B-6procedure checklist 6-22the Comparative C T Method 6-4the Standard Curve Method 6-4troubleshooting 8-12removingplate documents from the plate queue 4-40steps from a method 3-21Rep readout (from the Real Time tab) 4-27replacinggripper finger pads 7-52sample block module 7-6replicate wells, use of 6-20reproducible limits 6-19resizing panes, views, and plots 2-21restacking plates 4-42restoringdatabase connection A-22RQ Manager Software 1-25RQ Min/Max Confidence 6-28runningbatches of plates 4-39, 4-41single plate 4-25Ssamplecalibrator 6-20sample barsabout 6-31Index-6Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

sample block locking bar 7-8sample block locking bolt 7-8sample block moduleabout 1-5cleaning sample block module wells 7-15contamination 8-7replacing 7-6sample namesadding to a plate document 3-28importing into a plate document A-2Sample readout (from the Real Time tab) 4-27sample volume setting 3-22saturation, signal 7-27savingplate documents 2-17, 4-24template files 3-24scrutinizing allele calls 5-16SDS 7900HT Document (*.sds) filesSee plate documentsSDS 7900HT Template Document (*.sdt) filesSee templatesSDS Enterprise Databaseabout 1-22command line tools 1-26RQ Manager Software 1-25saving plate documents 4-24saving results 5-19, 6-53saving templates 3-25SDS Database 1-26SDS User Account Manager 1-27SNP Manager Software 1-25study-centric design 1-27SDS softwareabout 1-14disconnecting 4-30installing 7-55launching 2-7learning to use the software 2-10re-connecting 4-30upgrading 7-55Sealerinstallation 4-14overview 4-14Sealer, see microfluidic card sealerSealingguidelines 4-19TaqMan Low Density Array 4-19trimming fill reservoirs after sealing 4-21selectingwells from the plate grid 2-24Service Name A-22setup table fileabout A-4example files A-4exporting A-17importing into a plate document A-2structure A-4signal saturation 7-27Slope readout (from the Standard Curve Plot) 6-13SNP Manager Software 1-25spectral calibration, See pure dye runsStage readout (from the Real Time tab) 4-27Standard Curve Method 6-4Standard Curve Plotabout 6-13exporting as a graphic file A-16exporting data as a text file A-16standardsquantifying for absolute quantification B-6selecting for absolute quantification B-6selecting for relative quantification B-6startingplate queue 4-43run from the SDS software 4-25State readout (from the Real Time tab) 4-27Status readout (from the Real Time tab) 4-27stepadding to a method 3-21Step readout (from the Real Time tab) 4-27stoppingplate queue 4-44run from the SDS software 4-28studies 1-28study-centric design 1-27SYBR Green 1 Dyeabout D-3System performance, verifyingRNase P card, preparing 7-32Ttable paneabout 2-29exporting A-16TaqMan fluorogenic probeabout D-2designing B-2 to B-3TaqMan Low Density Arraycentrifuging 4-16components 4-10handling 4-14how it works 4-10loading 4-14loading guidelines 4-14number of targets per well 4-10overview 4-10placing in bucket 4-16quality control 4-12removing from packaging 4-14sealing 4-19Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideIndex-7DRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

single-plex TaqMan reagents 4-11TaqMan primers and probes 4-10trimming after sealing 4-21TaqMan reagents, description 4-10TaqMan RNase P Instrument Verification Platesabout 7-30analyzing 7-34kits C-5preparing a plate document 7-33running 7-30, 7-32Targets, mRNA, number per well 4-10tasks, See detector taskstemplatesabout 2-12creating a single plate document 3-26creating multiple plate documents 4-36saving as 3-24thermal cycler block, See sample block modulethermal cycling protocol, See methodsthresholdabout D-6configuring for automatic analysis 6-11improper setting 8-5threshold cyclecalculation D-6relationship to PCR product D-7Time readout (from the Real Time tab) 4-27Time Remaining readout (from the Real Timetab) 4-27T m , See melting temperaturetroubleshooting 8-2 to 8-207900HT instrument 8-14background runs 8-9chemistry problems 8-5 to 8-8computer 8-14end-point runs 8-13fixed-position bar code reader 8-17, 8-20pure dye runs 8-11real-time runs 8-12SDS software 8-14Zymark Twister Microplate Handler 8-17, 8-20turning ON the Applied Biosystems 7900HT Fast Real-Time PCR System 2-4Wresizing 2-21, 2-22showing 2-21warranty statement E-1well inspector 2-31YY Inter readout (from the Standard Curve Plot) 6-13Zzoomingallelic discrimination plot 5-14plate grid wells 2-26Zymark Twister Microplate HandlerSee Plate HandlerZymark Twister Softwareabout 1-14aligning the Plate Handler 7-41 to 7-48closing 7-48launchingUupgradingoperating system software 7-55SDS software 7-55Vviewing, well information 2-23viewshiding 2-21maximizing/minimizing 2-22Index-8Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:38 am, 4351684AIX.fm

Worldwide Sales and SupportApplied Biosystems vast distribution andservice network, composed of highly trainedsupport and applications personnel,reaches 150 countries on six continents.For sales office locations and technical support,please call our local office or refer to ourWeb site at www.appliedbiosystems.com.Applera is committed to providing theworld’s leading technology and informationfor life scientists. Applera Corporationconsists of the Applied Biosystems andCelera Genomics businesses.Headquarters850 Lincoln Centre DriveFoster City, CA 94404 USAPhone: +1 650.638.5800Toll Free (In North America): +1 800.345.5224Fax: +1 650.638.588409/2004www.appliedbiosystems.comPart Number 4351684 Rev. A